Multipart reagents having increased avidity for polymerase binding

ABSTRACT

Multivalent binding compositions including a particle-nucleotide conjugate having a plurality of copies of a nucleotide attached to the particle are described. The multivalent binding compositions allow one to localize detectable signals to active regions of biochemical interaction, e.g., sites of protein-protein interaction, protein-nucleic acid interaction, nucleic acid hybridization, or enzymatic reaction, and can be used to identify sites of base incorporation in elongating nucleic acid chains during polymerase reactions and to provide improved base discrimination for sequencing and array based applications.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/936,121, filed Jul. 22, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/579,794, filed on Sep. 23, 2019, which claimsthe benefit of U.S. Provisional Application No. 62/897,172 filed on Sep.6, 2019.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 15, 2020, isnamed 52933_733_501_SL.txt and is 4,337 bytes in size.

BACKGROUND

This disclosure herein relates to the field of molecular biology, suchas compositions, methods, and systems for nucleic acid hybridization andnucleic acid sequencing. In particular, it relates to compositions andmethods for nucleic acid hybridization to nucleic acid molecules coupledto a surface and sequencing of those nucleic acid molecules.

Nucleic acid hybridization protocols constitute an important part ofmany different nucleic acid amplification and analysis techniques. Thelimited specificity and reaction rates achieved through the use ofexisting nucleic acid hybridization protocols can have detrimentaleffects on the throughput and accuracy of downstream nucleic acidanalysis methods. Methods of stringency control often involve conditionscausing a significant decrease in the number of hybridized complexes.Therefore, there is a need for an improved method to achieve a highstringency of hybridization during the sequencing analysis.

Nucleic acid sequencing can be used to obtain information in a widevariety of biomedical contexts, including diagnostics, prognostics,biotechnology, and forensic biology. Various sequencing methods havebeen developed including Maxam-Gilbert sequencing and chain-terminationmethods, or de novo sequencing methods including shotgun sequencing andbridge PCR, or next-generation methods including polony sequencing, 454pyrosequencing, Illumina sequencing, SOLiD sequencing, Ion Torrentsemiconductor sequencing, HeliScope single molecule sequencing, SMRT®sequencing, and others. Despite advances in DNA sequencing, manychallenges to cost effective, high throughput sequencing remainunaddressed. The present disclosure provides novel solutions andapproaches to addressing many of the shortcomings of existingtechnologies.

SUMMARY

Provided herein are methods of determining the identity of a nucleotidein a target nucleic acid comprising: (a) providing a compositioncomprising: (i) a target nucleic acid comprising two or more repeats ofan identical sequence; (ii) two or more primer nucleic acidscomplementary to one or more regions of said target nucleic acid; and(iii) two or more polymerase molecules; (b) contacting said compositionwith a multivalent binding composition comprising a polymer-nucleotideconjugate under conditions sufficient to allow a binding complex to beformed between said polymer-nucleotide conjugate and the composition ofstep (a), wherein the polymer-nucleotide conjugate comprises two or morecopies of a nucleotide and optionally one or more detectable labels; and(d) detecting said binding complex, thereby establishing the identity ofsaid nucleotide in the target nucleic acid. In some embodiments, thetarget nucleic acid is DNA. In some embodiments, the detection of thebinding complex is performed in the absence of unbound or solution-bornepolymer nucleotide conjugates. In some embodiments, the target nucleicacid has been replicated or amplified or has been produced byreplication or amplification. In some embodiments, the detectable labelis a fluorescent label. In some embodiments, detecting the complexcomprises a fluorescence measurement. In some embodiments, themultivalent binding composition comprises one type of polymer-nucleotideconjugate. In some embodiments, the multivalent binding compositioncomprises two or more types of polymer-nucleotide conjugates. In someembodiments, each type of the two or more types of polymer-nucleotideconjugates comprises a different type of nucleotide. In someembodiments, the multivalent binding composition consists of three typesof polymer-nucleotide conjugates and wherein each type of the threetypes of polymer-nucleotide conjugates comprises a different type ofnucleotide. In some embodiments, the binding complex further comprises ablocked nucleotide. In some embodiments, the blocked nucleotide is a3′-O-azidomethyl, 3′-O-methyl nucleotide, or 3′-O-alkyl hydroxylamine.In some embodiments, said contacting occurs in the presence of an ionthat stabilizes said binding complex, said complex comprising a polymernucleotide conjugate, two or more polymerase molecules, and two or morebinding sites within the target nucleic acid. In some embodiments, thecontacting is done in the presence of strontium, magnesium, calciumions, or any combination thereof. In some embodiments, the polymerasemolecule is catalytically inactive. In some embodiments, the bindingcomplex has a persistence time of greater than 2 seconds. In someembodiments, methods further comprise hybridizing the two or more primernucleic acids to the one or more regions of said target nucleic acid bybringing the two or more primer nucleic acids into contact with ahybridizing composition comprising said target nucleic acid at aconcentration of 1 nanomolar or less under conditions sufficient forsaid target nucleic acid to hybridize to the two or more primer nucleicacids in 30 minutes or less. In some embodiments, the two or more primernucleic acids are coupled to a hydrophilic polymer surface having awater contact angle of less than 45 degrees. In some embodiments, thehybridization composition further comprises: (a) at least one organicsolvent having a dielectric constant of no greater than about 115 asmeasured at 68 degrees Fahrenheit; and (b) a pH buffer. In someembodiments, the hybridization composition further comprises: (a) atleast one organic solvent that is polar and aprotic; and (b) a pHbuffer.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entirety tothe same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety. In the event of a conflictbetween a term herein and a term in an incorporated reference, the termherein controls.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some novel features of the methods and compositions disclosed herein areset forth in the present disclosure. A better understanding of thefeatures and advantages of the methods and compositions disclosed hereinwill be obtained by reference to the following detailed description thatsets forth illustrative embodiments, in which the principles of thedisclosed compositions and methods are utilized, and the accompanyingdrawings of which:

FIGS. 1A-B provide non-limiting examples of image data that demonstratethe improvements in hybridization stringency, speed, and efficacy thatmay be achieved through the reformulation of the hybridization bufferused for solid-phase nucleic acid amplification, as described herein.FIG. 1A provides examples of image data for two different hybridizationbuffer formulations and protocols. FIG. 1B provides an example of thecorresponding image data obtained using a standard hybridization bufferand protocol.

FIG. 2 illustrates a workflow for nucleic acid sequencing using thedisclosed hybridization methods on low binding surfaces and non-limitingexamples of the processing times that may be achieved.

FIG. 3 shows the surface template hybridization images (NASA results at100 pM) of the samples corresponding to the compositions used forhybridization.

FIG. 4 shows a table with hybridization design of experiment spotcounts.

FIG. 5 shows the post nucleic acid surface amplification PCR images ofthe samples.

FIG. 6 shows a work flow according to various embodiments disclosedherein.

FIG. 7 shows a work flow for a sequence reaction according to variousembodiments described herein.

FIG. 8 shows a sample nucleic acid hybridization workflow according tovarious embodiments described herein.

FIG. 9A-9B show how sample nucleic acids hybridized to the nucleic acidmolecules coupled to the low-non-specific binding surface is visualized(FIG. 9A) or amplified (FIG. 9B) according to various embodimentsdescribed herein.

FIG. 10 schematically depicts an example computer control system.

FIG. 11 shows a workflow of purification and isolation of sample nucleicacids from a biological sample, library preparation, and hybridizationaccording to various embodiments described herein.

FIGS. 12A-12H illustrate the steps for sequencing a target nucleic acidutilizing a non-limiting example of a multivalent binding composition:FIG. 12A illustrates non-limiting example 8 of attaching a targetnucleic acid to a surface; FIG. 12B illustrates a non-limiting exampleof clonally amplifying the target nucleic acid to form clusters ofamplified target nucleic acid molecules; FIG. 12C illustrates anon-limiting example of priming the target nucleic acid to produce aprimed target nucleic acid; FIG. 12D illustrates a non-limiting exampleof contacting the primed target nucleic acid with the multivalentbinding composition and polymerase to form a binding complex; FIG. 12Eillustrates a non-limiting example of the images of the binding complexcaptured on the surface; FIG. 12F illustrates a non-limiting example ofextending a primer strand by one nucleotide; FIG. 12G illustrates anon-limiting example of another cycle of contacting the primed targetnucleic acid to the multivalent binding composition and polymerase toform a binding complex; and FIG. 12H illustrates non-limiting examplesof images of a binding complex captured on the surface in subsequentsequencing cycles.

FIG. 13 shows a flow chart outlining steps for sequencing a targetnucleic acid and extending a primer strand through a single baseaddition according to various embodiments described herein.

FIG. 14 shows a flow chart outlining steps for sequencing a targetnucleic acid and extending a primer strand through incorporating thenucleotide on the particle-nucleotide conjugate according to variousembodiments described herein.

FIGS. 15A-15B illustrate a non-limiting example of detecting a targetnucleic acid using the polymer-nucleotide conjugates. FIG. 15A shows thestep of contacting the polymerase and polymer-nucleotide conjugates tosome nucleic acid molecules; FIG. 15B shows the binding complex formedbetween the polymerase, polymer-nucleotide conjugates, and the targetnucleic acid molecules.

FIGS. 16A-16C show schematic representations of non-limiting examples ofvarying configurations of the polymer-nucleotide conjugates: FIG. 16Ashows polymer-nucleotide conjugates having various multi-armconfigurations; FIG. 16B shows a polymer-nucleotide conjugate having thepolymer branch radiating from the center; and FIG. 16C showspolymer-nucleotide conjugates having the binding moiety biotin.

FIG. 17 shows a generalized graphical depiction of the increase insignal intensity that has been observed during binding, persistence, andwashing and removal of multivalent substrates.

FIGS. 18A-18J show fluorescence images of the steps in a sequencingreaction using multivalent PEG-substrate compositions. FIG. 18A. showsred and green fluorescent images post exposure of DNA RCA templates (Gand A first base) to 500 nM base labeled nucleotides (A-Cy3 and G-Cy5)in exposure buffer containing 20 nM Klenow polymerase and 2.5 mM Sr+2.Images were collected after washing with imaging buffer with the samecomposition as the exposure buffer but containing no nucleotides orpolymerase. Contrast was scaled to maximize visualization of the dimmestsignals, but no signals persisted following washing with imaging buffer(FIG. 18A, inset). FIGS. 18B-18 show fluorescence images showingmultivalent PEG-nucleotide (base-labeled) ligands PB1 (FIG. 18B), PB2(FIG. 18C), PB3 (FIG. 18D), and PB5 (FIG. 18E) having an effectivenucleotide concentration of 500 nM after mixing in the exposure bufferand imaging in the imaging buffer as described above. FIG. 18F showsfluorescence image showing multivalent PEG-nucleotide (base-labeled)ligand PB5 at 2.5 μM after mixing in the exposure buffer and imaging inthe imaging buffer as above. FIGS. 18G-18I show fluorescence imagesshowing further base discrimination by exposure of the multivalentbinding composition to inactive mutants of klenow polymerase (FIG. 18G.D882H; FIG. 18H. D882E; FIG. 18I. D882A) vs. the wild type Klenow(control) enzyme (FIG. 18J).

FIGS. 19A-19B show the efficacy of the multivalent reporter compositionsin determining the base sequence of a DNA sequence over 5 sequencingcycles: FIG. 19A shows images and expected sequences for templates takenafter each sequencing cycle; and FIG. 19B shows aligned sequencingresults utilizing the images taken in FIG. 19A. FIG. 19B discloses SEQID NOS: 5-7, 5-6, 8-14, 5, 13, 5 and 15, respectively, in order ofappearance.

FIGS. 20A-20G show fluorescence images of multivalent polyethyleneglycol (PEG) polymer-nucleotide (base-labeled) conjugates having aneffective nucleotide concentration of 500 nM and varying PEG branchlength, after contacting to a support surface comprising DNA templates(comprising G or A as the first base; prepared using rolling circleamplification (RCA)) in an exposure buffer comprising 20 nM Klenowpolymerase and 2.5 mM Sr+2. Images were acquired after washing with animaging buffer having the same composition as the exposure buffer butlacking nucleotides and polymerase. Panels show images obtained usingmultivalent PEG-nucleotide ligands with arm lengths as follows: FIG.20A: 1K PEG. FIG. 20B: 2K PEG. FIG. 20C: 3K PEG. FIG. 20D: 5K PEG. FIG.20E: 10K PEG. FIG. 20F: 20K PEG. FIG. 20G shows images obtained using10K PEG and an inactive klenow polymerase comprising the mutation D882H.

FIG. 21 shows a quantitative representation of the fluorescenceintensities in the images shown in FIGS. 20A-20F, separated by colorvalue, with orange trace corresponding to the red label (Cy3 label; Abases) and blue trace corresponding to the green label (Cy5 label; Gbases).

FIG. 22 shows normalized fluorescence from multivalent substrates boundto DNA clusters as described for FIGS. 18A-18J, with the substratecomplexes formed in the presence (condition B) and absence (condition A)of Triton-X100 (0.016%).

FIGS. 23A-23B show plots of normalized fluorescence intensity measuredfor multivalent polymer-nucleotide conjugates and free nucleotides. FIG.23A shows two replicates of a multivalent polymer-nucleotide conjugatebound to DNA clusters (Conditions A and B) vs. binding complexes formedusing labeled free nucleotides (Condition C) after 1 minute. FIG. 23Bshows the time course of fluorescence from multivalent substratecomplexes over the course of 60 min.

DETAILED DESCRIPTION

Disclosed herein are methods, compositions, systems, and kits fornucleic acid hybridization to nucleic acid molecules coupled to asurface. The methods, compositions, systems, and kits described hereinare particularly useful for nucleic acid amplification, nucleic acidsequencing, or a combination thereof. The methods, compositions,systems, and kits described herein enable superior nucleic acidhybridization performance. For example, nucleic acid hybridizationaccording to the methods, compositions, systems, and kits describedherein can be performed for a fraction of the cost and/or in a fractionof the time as compared to nucleic acid hybridization methods requiringhigh temperature (e.g., 90 degrees Celsius) incubations, long incubationtimes (e.g., 1-2 hours), and large amounts of input nucleic acid (e.g.,10 nanomolar). This is accomplished by utilizing optimized hybridizationcompositions (e.g., buffers, organic solvents) in combination with lownon-specific binding surfaces that are hydrophilic.

Nucleic acid hybridization methods requiring high temperatureincubations, long incubation times, and large amounts of input nucleicacid are complex, time consuming, and lack the specificity andefficiency needed for cost-effective high throughput applications. Atleast one reason such nucleic acid hybridization methods lackspecificity and efficiency is that the surfaces used are prone tonon-specific binding of proteins or nucleic acids, contributing toincreased background signal.

The methods, compositions, systems, and kits described herein providesuperior hybridization specificity and efficiency of target nucleic acidmolecules to surface-bound nucleic acid molecules, as compared tonucleic acid hybridization methods using surfaces prone to non-specificbinding reactions. Described herein, are methods and systems utilizing alow non-specific binding surface, thereby reducing background signal.The low non-specific binding surfaces described herein are engineered sothat proteins, nucleic acids, and other biomolecules do not “stick” tothe substrate of the surface. The low non-specific binding surfacesdescribed herein are hydrophilic. In some instances, the lownon-specific binding surfaces have a water contact angle of less than orequal to about 50 degrees.

The methods described herein comprise hybridizing a target nucleic acidto a nucleic acid molecule coupled to a hydrophilic surface (e.g., a lownon-specific binding surface) in the presence of the hybridizationcompositions described herein. The methods described herein are usefulfor nucleic acid hybridization, amplification, sequencing, or acombination thereof. In some instances, the methods described hereinachieve superior hybridization performance on a low non-specific bindingsurface. In addition, in some instances, the methods described hereinachieve a non-specific cyanine dye-3 (Cy3) dye absorption of less thanabout 0.25 molecules/μm².

Optimized hybridization compositions described herein, for example, whenused with low non-specific binding surfaces, enable isothermalhybridization reactions to be performed at 60 degrees Celsius for as fewas 2 minutes, using as little as 50 picomolar concentration of inputnucleic acid. Methods described herein provide (i) superiorhybridization rates, (ii) superior hybridization specificity, (iii)superior hybridization stringency, (iv) superior hybridizationefficiency (or yield), (v) reduced requirements for the amount ofstarting material necessary, (vi) lowered temperature requirements forisothermal or thermal ramping amplification protocols, (vii) increasedannealing rates, and (viii) a yield having a low percentage of the totalnumber target nucleic acid molecules (or amplified clusters of targetnucleic acid molecules) being associated with the surface withouthybridizing to the surface bound nucleic acid, as compared tohybridization reactions using non-specific binding surfaces. Theincreased performance and reduced cost and time required to perform ahybridization reaction make the methods, compositions, systems, and kitsdescribed herein ideally suited for high throughput nucleic acidhybridization, amplification, and sequencing applications.

Hybridization formulations using, for example, saline sodium citratebuffer achieve poor hybridization specificity or efficiency when usedwith hybridization protocols using the non-specific binding surfacesdescribed herein. The hybridization reaction or annealing interactionbetween target nucleic acid molecules in the solution and nucleic acidmolecules coupled to the low non-specific binding surfaces can beimpacted by several factors, including the availability of hydrogenbonding partners in the solution and the polarity of the solution. Ingeneral, nucleic acids preferentially inhabit bulk solution wherepossible in order to take advantage of the additional entropicstabilization presented by the ability to access dynamic states inthree, rather than two, dimensions such as would be available on a solidsurface. At equilibrium, in a system comprising a nucleic acid, asolution, and a hydrophilic surface (e.g., low non-specific bindingsurface), a nucleic acid molecule will be preferentially stabilized insolution, rather than in a surface-bound state when the solvent isaqueous.

Hybridization compositions and methods utilizing protic solvents (e.g.,saline sodium citrate buffer) are disadvantageous for nucleic acidhybridization reactions with the low non-specific binding surfacesdescribed herein, because aprotic solvents provide a favorableenvironment for the target nucleic acid molecules to stay in solution,rather than binding to the low non-specific binding surface. This is dueto the ability of the protic solvent to provide sufficient hydrogenbonding partners of sufficient size and distribution such that hydrogenbonding interactions between the exposed hydrogen bond donors andacceptors along the nucleic acid backbone, or, any exposed sidechainmoieties, occur.

By contrast, the hybridization compositions described herein drive thetarget nucleic acid molecule to the low non-specific binding surfacewhile in solution, by utilizing an aprotic organic solvent, such as, forexample, formamide. The aprotic solvents described herein reduce theproportion of solvent molecules capable of satisfying the hydrogenbonding requirements of the nucleic acid chain, and make it possible tocreate an entropic penalty in the bulk solution, which will drive thesystem toward stabilization by depositing the nucleic acid on thesurface (e.g., the entropic penalty caused by ordering the bulk solutionto accommodate the unbonded hydrogen bonding elements in the nucleicacid becomes greater than the entropic penalty caused by loss of thethird dimension of dynamic freedom when the polymer is adsorbed to thesurface). Furthermore, introduction of an aprotic organic solvent intothe solution may help drive down the entropy and in turn provides a morefavorable environment for the nucleic acid to bind to the hydrophilicsurface. For example, addition of aprotic solvent acetonitrile helps todrive the nucleic acid in the solution towards a surface bound state.

The hybridization compositions described herein may further compriseconcentrations of protic and aprotic organic solvents, in order toprevent precipitation of the target nucleic acid from solution that canbe caused by high concentrations of aprotic solvent in the solution. Inthis manner, hybridization compositions described herein may cause thenucleic acids to selectively associate with hydrophilic surfaces (e.g.,low non-specific binding surfaces), while remaining substantiallysolvated.

The hybridization compositions described herein, may comprise crowdingagents, which are capable of modulating interactions of nucleic acidswith the bulk solution. In some instances, the hybridizationcompositions comprise relaxing agents, divalent cations, orintercalating agents, which are capable of modulating the dynamics ofthe polymer itself and may also modulate the interactions of nucleicacids with surfaces in the presence of partially aprotic bulk solvents.Providing such agents in combination with buffers containing somefraction of aprotic or non-hydrogen-bonding components can, in someinstances, provide superior control over the interaction of nucleic acidmolecules with hydrophilic surfaces.

Various aspects of the disclosed nucleic acid hybridization methods maybe applied to solution-phase or solid-phase nucleic acid hybridization,and also to any other type of nucleic acid amplification, or, analysisapplications (e.g., nucleic acid sequencing), or any combinationthereof. It shall be understood that different aspects of the disclosedmethods, devices, and systems can be appreciated individually,collectively, or in combination with each other.

The methods, compositions, systems, and kits described herein are usefulfor a wide range of applications beyond those involving nucleicacid-surface interactions, because the same thermodynamic parametersoptimized by the methods and compositions described herein govern anumber of interactions between polymers and biomolecules, as well aspolymer and surface interactions and biomolecule and surfaceinteractions. Thus, the methods compositions, systems and kits describedherein may be applied to tune the polarity, or the hydrogen bondingpotential, or a combination thereof, of a solvent in other systemsinvolving these interactions.

Solution-based hybridization is the foundation for many solution-basedmolecular biology and solution-phase DNA manipulation applications, mostnotably the polymerase chain reaction (PCR) (L. Garibyan and N. Avashia,J. Invest. Dermatol., 2013, 133, e6; Z. Xiao, D. Shangguan, Z. Cao, X.Fang, and W. Tan, 2008, DNA guided drug delivery, Chemistry 14, 1769-75;and F. Wei, C. Chen, L. Zhai, N. Zhang, and X. S. Zhao, 2005, DNA basedbiosensors, J. Am. Chem. Soc., 127, 5306-5307; and S. Tyagi and F. R.Kramer, Nat. Biotechnol., 1996, 14, 303-308). The diffusion rates inmany of these reactions are sufficient to drive efficient hybridizationand the formation of a functional double-stranded form, which can beanalyzed kinetically as a second order kinetic reaction, whereby theforward reaction of duplex formation is second order and the reversereaction comprising the dissociation of the duplex structure to form thetwo single stranded complements (strands A and B) is first order (Han,C., Improvement of the Speed and Sensitivity of DNA Hybridization UsingIsotachophoresis, Stanford Thesis. 2015). These reactions may be writtenas:

$A + {B\begin{matrix}\overset{k_{on}}{\rightarrow} \\\underset{k_{off}}{\leftarrow}\end{matrix}{AB}}$$\frac{d\lbrack{AB}\rbrack}{dt} = {{{k_{on}\lbrack A\rbrack}\lbrack B\rbrack} - {k_{off}\lbrack{AB}\rbrack}}$

Various approaches have been deployed to increase not only the speed ofthe hybridization reaction but also the reaction specificity in the wakeof confounding DNA non-complementary fragments. Such approaches include,but are not limited to, the addition of MgCl₂ and higher saltconcentrations, and lower temperatures to accelerate the reactions (H.Kuhn, V. V Demidov, J. M. Coull, M. J. Fiandaca, B. D. Gildea, and M. D.Frank-Kamenetskii, J. Am. Chem. Soc., 2002, 124, 1097-1103; N. A. Strausand T. I. Bonner, Biochim. Biophys. Acta, Nucleic Acids Protein Synth.,1972, 277, 87-95). The trade-off for accelerated reaction rates is oftenreaction specificity (J. M. S. Bartlett and D. Stirling, PCR protocols,Humana Press, 2003; W. Rychlik, W. J. Spencer, and R. E. Rhoads, NucleicAcids Res., 1990, 18). Additional methods are sometimes employed thatyield potential improvements of reaction specificity through the use ofvolume exclusion, or, molecular crowding techniques, or a combinationthereof that utilize inert polymers as hybridization buffer additives(R. Wieder and J. G. Wetmur, Biopolymers, 1981, 20, 1537-1547, J. G.Wetmur, Biopolymers, 1975, 14, 2517-2524). In addition, organic solventshave been employed as additives to accelerate hybridization kinetics andmaintain reaction specificity (N. Dave and J. Liu, J. Phys. Chem. B,2010, 114, 15694-15699).

While hybridization improvements in solution may be translated tosurface-based hybridization techniques, surface-based hybridizationneeds have far ranging implications for many critical bioassays, such asgene expression analysis (D. T. Ross, U. Scherf, M. B. Eisen, C. M.Perou, C. Rees, P. Spellman, V. Iyer, S. S. Jeffrey, M. Van de Rijn, M.Waltham, A. Pergamenschikov, J. C. Lee, D. Lashkari, D. Shalon, T. G.Myers, J. N. Weinstein, D. Botstein, and P. O. Brown, Nat. Genet., 2000,24, 227-235; A. Adomas, G. Heller, A. Olson, J. Osborne, M. Karlsson, J.Nahalkova, L. Van Zyl, R. Sederoff, J. Stenlid, R. Finlay, and F. O.Asiegbu, Tree Physiol., 2008, 28, 885-897; M. Schena, D. Shalon, R. W.Davis, and P. O. Brown, Science, 1995, 270, 467-470), diagnosis ofdisease (J. Marx, Science, 2000, 289, 1670-1672), genotyping and SNPdetection (J. G. Hacia, J. B. Fan, O. Ryder, L. Jin, K. Edgemon, G.Ghandour, R. A. Mayer, B. Sun, L. Hsie, C. M. Robbins, L. C. Brody, D.Wang, E. S. Lander, R. Lipshutz, S. P. Fodor, and F. S. Collins, Nat.Genet., 1999, 22, 164-167), rapid nucleic acid based pathogen screening,next generation sequencing (NGS) and a host of other genomics basedapplications (M. J. Heller, Annu. Rev. Biomed. Eng., 2002, 4, 129-53).The common necessity of all of these reactions is high reactionspecificity in a highly multiplexed solution of target sequences thatmay range from thousands to billions of different sequences, such thatthe targets are quickly tethered on a solid surface for subsequentprobing, or, amplification, or a combination thereof to enable DNA (orother nucleic acid) interrogation for applications such as sequencing orarray-based analysis. The efficiencies of surface-based hybridizationreactions were found to be much less than that of in solution reactions,e.g., about an order of magnitude less efficient. A great deal of workhas been done in past attempts to create a hybridization method forsolid surface that provides high specificity and acceleratedhybridization reaction rates (D. Y. Zhang, S. X. Chen, and P. Yin, Nat.Chem., 2012, 4, 208-14).

Disclosed herein are innovative combinations of approaches gleaned fromstudies of surface- and solution-based hybridization as outlined above,as well as from other fields of study that include DNA hydration andquadruplex studies, which lead to substantial improvements inhybridization kinetics and specificity. The disclosed hybridizationcompositions provide for highly specific (e.g., >2 orders of magnitudeimprovement over traditional approaches) and accelerated hybridization(e.g., >1-2 orders of magnitude improvement over traditional approaches)when used with low non-specific binding surfaces for applications suchas next generation sequencing (NGS) and other bioassays that requirehighly specific nucleic acid hybridization in a multiplexed poolcomprised of a large number of target sequences.

Hybridization Methods

Provided herein are methods for nucleic acid hybridization between asample nucleic acid molecule and a capture nucleic acid molecule.Referring to FIG. 11, the sample nucleic acid molecule is isolated andpurified from a biological sample obtained from a subject 1110. Alibrary of isolated and purified sample nucleic acid molecules isprepared 1111. The library of sample nucleic acid molecules ishybridized to nucleic acid molecules coupled to a low non-specificbinding surface described herein in the presence of a hybridizationcomposition 1112.

Biological Sample. The biological samples disclosed herein may comprisenucleic acid molecules, amino acids, polypeptides, proteins,carbohydrates, fats, or viruses. In an example, a biological sample is anucleic acid sample including one or more nucleic acid molecules.Exemplary biological samples may include polynucleotides, nucleic acids,oligonucleotides, cell-free nucleic acid (e.g., cell-free DNA (cfDNA)),circulating cell-free nucleic acid, circulating tumor nucleic acid(e.g., circulating tumor DNA (ctDNA)), circulating tumor cell (CTC)nucleic acids, nucleic acid fragments, nucleotides, DNA, RNA, peptidepolynucleotides, complementary DNA (cDNA), double stranded DNA (dsDNA),single stranded DNA (ssDNA), plasmid DNA, cosmid DNA, chromosomal DNA,genomic DNA (gDNA), viral DNA, bacterial DNA, mtDNA (mitochondrial DNA),ribosomal RNA, cell-free DNA, cell free fetal DNA (cffDNA), mRNA, rRNA,tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, viral RNA,and the like.

Any substance that comprises nucleic acid may be the source of thebiological sample. The substance may be a fluid, e.g., a biologicalfluid. A fluidic substance may include, but is not limited to, blood,cord blood, saliva, urine, sweat, serum, semen, vaginal fluid, gastricand digestive fluid, spinal fluid, placental fluid, cavity fluid, ocularfluid, serum, breast milk, lymphatic fluid, or combinations thereof. Thesubstance may be solid, for example, a biological tissue. The substancemay comprise normal healthy tissues, diseased tissues, or a mix ofhealthy and diseased tissues.

Biological samples described herein are obtained from various subjects.A subject may be a living subject or a dead subject. Examples ofsubjects may include, but are not limited to, humans, mammals, non-humanmammals, rodents, amphibians, reptiles, canines, felines, bovines,equines, goats, ovines, hens, avines, mice, rabbits, insects, slugs,microbes, bacteria, parasites, or fish. The subject, in some cases, is apatient who is having, suspected of having, or at a risk of developing adisease or disorder. In some instances, the subject may be a pregnantwoman. In some instances, the subject may be a normal healthy pregnantwoman. In some instances, the subject may be a pregnant woman who is ata risking of carrying a baby with certain birth defect.

A sample may be obtained from a subject by various approaches. Forexample, a sample may be obtained from a subject through accessing thecirculatory system (e.g., intravenously or intra-arterially via asyringe or other apparatus), collecting a secreted biological sample(e.g., saliva, sputum urine, feces), surgically (e.g., biopsy) acquiringa biological sample (e.g., intra-operative samples, post-surgicalsamples), swabbing (e.g., buccal swab, oropharyngeal swab), orpipetting.

Biological Sample Processing. The biological sample described herein, insome instances, is processed. Processing comprises filtering a sample,binding a component of the sample that contains an analyte, binding theanalyte, stabilizing the analyte, purifying the analyte, or acombination thereof. Non-limiting examples of sample components arecells, viral particles, bacterial particles, exosomes, and nucleosomes.In some instances, blood plasma or serum is isolated from a whole bloodsample. In some instances, the whole blood is obtained from venous bloodor capillary blood of a subject described herein.

Library Preparation of Sample Nucleic Acids. The sample nucleic acidsdescribed herein, in some cases, are converted to a library by labelingthe sample nucleic acids with a label, barcode or tag. The library ofsample nucleic acids are amplified in some instances, for example, byisothermal amplification. Non-limiting examples of amplification methodsinclude, but are not limited to, loop mediated isothermal amplification(LAMP), nucleic acid sequence based amplification (NASBA), stranddisplacement amplification (SDA), multiple displacement amplification(MDA), rolling circle amplification (RCA), ligase chain reaction (LCR),helicase dependent amplification (HDA), nicking enzyme amplificationreaction (NEAR), recombinase polymerase amplification (RPA), andramification amplification method (RAM).

In some instances, isothermal amplification is used. In some instances,amplification is isothermal with the exception of an initial heatingstep before isothermal amplification begins. A number of isothermalamplification methods, each having different considerations andproviding different advantages, are known in the art and have beendiscussed in the literature, e.g., by Zanoli and Spoto, 2013,“Isothermal Amplification Methods for the Detection of Nucleic Acids inMicrofluidic Devices,” Biosensors 3: 18-43, and Fakruddin, et al., 2013,“Alternative Methods of Polymerase Chain Reaction (PCR),” Journal ofPharmacy and Bioallied Sciences 5(4): 245-252, each incorporated hereinby reference in its entirety.

In some instances, the amplification method is Rolling CircleAmplification (RCA). RCA is an isothermal nucleic acid amplificationmethod which allows amplification of the probe DNA sequences by morethan 10⁹-fold at a single temperature, typically about 30° C. Numerousrounds of isothermal enzymatic synthesis are carried out by 029 DNApolymerase, which extends a circle-hybridized primer by continuouslyprogressing around the circular DNA probe. In some instances, theamplification reaction is carried out using RCA, at about 28° C. toabout 32° C. Suitable methods of RCA are described in U.S. Pat. No.6,558,928.

In some instances, amplifying comprises targeted amplification. In someinstances, amplifying a nucleic acid comprises contacting a nucleic acidwith at least one primer having a sequence corresponding to a targetchromosome sequence. Amplification may be multiplexed, involvingcontacting the nucleic acid with multiple sets of primers, wherein eachof a first pair in a first set and each of a pair in a second set areall different.

Hybridization. Methods described herein comprise bringing a samplenucleic acid molecule into contact with a capture nucleic acid moleculethat is optionally coupled to a low non-specific binding surface in thepresence of a hybridization composition described herein. In some cases,the capture nucleic acid molecule is coupled to the low non-specificbinding surface and hybridization occurs on the surface. In some cases,the capture nucleic acid molecules are not coupled to the lownon-specific binding surface and hybridization occurs in solution.Methods provided herein further comprising hybridizing the samplenucleic acid molecule with the capture nucleic acid molecule.

Methods described herein comprise hybridizing at least a portion of thesample nucleic acid molecule comprising a nucleic acid sequence that issufficiently complementary to a portion of the capture nucleic acidmolecule. The portion of the capture nucleic acid molecule and thesample nucleic acid molecule can be at least or equal to about 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50 nucleotides. The portion of the capturenucleic acid molecule and the sample nucleic acid molecule can bebetween 4 and 50, 5 and 49, 6 and 48, 7 and 47, 8 and 46, 9 and 45, 10and 44, 11 and 43, 12 and 42, 13 and 41, 14 and 40, 15 and 39, 16 and38, 17 and 37, 18 and 36, 19 and 35, 20 and 34, 21 and 33, 22 and 32, 23and 31, 24 and 30, 25 and 29, 26 and 28 nucleotides. The portion of thecapture nucleic acid molecule and the sample nucleic acid molecule canbe between 8 and 20 nucleotides. In some instances, at least 90% of thenucleic acids in the portion of the sample nucleic acid molecule and theportion of the capture nucleic acid molecule hybridize completely. Insome instances, at least 95% of the nucleic acids in the portion of thesample nucleic acid molecule and the portion of the capture nucleic acidmolecule hybridize completely. In some instances, between 95-100% of thenucleic acids in the portion of the sample nucleic acid molecule and theportion of the capture nucleic acid molecule hybridize completely.

A non-limiting example provided in FIG. 8 shows one or more samplenucleic acid molecules 801 to be circularized 802 using ligation (e.g.,splint ligation) 802, and introduced to one or more nucleic acidmolecules 808 coupled to a hydrophilic substrate 807 of a lownon-specific binding surface 806 in the presence of a hybridizationcomposition 805. In this example, the low-non-specific binding surfaceis submerged in the hybridization composition. In another example, theone or more sample nucleic acid molecules is introduced to thehybridization composition before introduction to the one or more nucleicacid molecules 808 coupled to the hydrophilic substrate 807 of the lownon-specific binding surface 806. Hybridization occurs between thesample nucleic acid molecule and the surface-coupled nucleic acidmolecule 809.

Sample Nucleic Acids. The one or more sample nucleic acid moleculesdescribed herein is derived from a biological sample described herein.The sample nucleic acid molecules may be a deoxyribonucleic acid (DNA)molecule or a ribonucleic acid (RNA) molecule. In some instances, theDNA is selected from cell-free DNA (cfDNA)), circulating cell-freenucleic acid, circulating tumor nucleic acid (e.g., circulating tumorDNA (ctDNA)), circulating tumor cell (CTC) nucleic acids, nucleic acidfragments, nucleotides, DNA, complementary DNA (cDNA), double strandedDNA (dsDNA), single stranded DNA (ssDNA), plasmid DNA, cosmid DNA,chromosomal DNA, genomic DNA (gDNA), viral DNA, bacterial DNA, and mtDNA(mitochondrial DNA). In some instances, the RNA is selected fromribosomal RNA, cell-free DNA, cell free fetal DNA (cffDNA), mRNA, rRNA,tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, viral RNA,and the like.

Coupling the Capture Nucleic Acids to the Surface. The nucleic acidmolecules coupled to the surface (e.g., capture molecules) may becoupled to the surface by a number of suitable options. In someinstances, the nucleic acid molecules are coupled to the surface throughcovalent bonds. In some instances, the nucleic acid molecules arecoupled to the surface through noncovalent bonds. In some instances, thenucleic acid molecules are attached to the surface through abio-interaction. Non-limiting examples of bio-interaction surfacechemistry include biotin-streptavidin interactions (or variationsthereof), polyhistidine (his) tag—Ni/NTA conjugation chemistries,methoxy ether conjugation chemistries, carboxylate conjugationchemistries, amine conjugation chemistries, NHS esters, maleimides,thiol, epoxy, azide, hydrazide, alkyne, isocyanate, and silane.

Compositions

Provided herein are hybridization compositions. The hybridizationcompositions of the present disclosure comprise at least one organicsolvent, which in some cases is polar and aprotic (e.g., having adielectric constant of less than or equal to about 115 as measured at 68degrees F.). The hybridization compositions comprise a pH buffer.Optionally, the hybridization compositions comprise one or moremolecular crowding/volume exclusion agents, one or more additives thatimpact DNA melting temperatures, one or more additives that impact DNAhydration, or any combination thereof. The hybridization compositionsdescribed herein can be used with the low non-specific binding surfacesdescribed herein, such as, for example, silicon dioxide coated with lowbinding polymers (e.g., polyethylene glycol (PEG)), for genotyping orsequencing related technologies. Genotyping and sequencing may beachieved using any or a combination of the following hybridizationcomposition components.

Organic Solvent: An organic solvent is a solvent or solvent systemcomprising carbon-based or carbon-containing substance capable ofdissolving or dispersing other substances. An organic solvent may bemiscible or immiscible with water.

Polar Solvent: A polar solvent, as included in the hybridizationcomposition described herein, is a solvent or solvent system comprisingone or more molecules characterized by the presence of a permanentdipole moment, e.g., a molecule having a spatially unequal distributionof charge density. A polar solvent may be characterized by a dielectricconstant of 20, 25, 30, 35, 40, 45, 50, 55, 60 or higher, or by a valueor a range of values incorporating any of the aforementioned values. Forexample, a polar solvent may have a dielectric constant of higher than100, higher than 110, higher than 111, or higher than 115. In somecases, the dielectric constant is measured at a temperature of greaterthan or equal to about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 degreesFahrenheit (F). In some cases, the dielectric constant is measured at atemperature of less than or equal to about −20, −25, −30, −35, −40, −45,−50, −55, −60, −65, −70, −75, −80, −85, −90, −95, −100, −150, −200,−250, −300, −350, −400, −450, or −459 degrees F. In some cases, thedielectric constant is measured at a temperature of at 68 degrees F. Insome cases, the dielectric constant is measured at a temperature of at20 degrees F.

A polar solvent as described herein may comprise a polar aproticsolvent. A polar aprotic solvent as described herein may further containno ionizable hydrogen in the molecule. In addition, polar solvents orpolar aprotic solvents may be preferably substituted in the context ofthe presently disclosed compositions with a strong polarizing functionalgroups such as nitrile, carbonyl, thiol, lactone, sulfone, sulfite, andcarbonate groups so that the underlying solvent molecules have a dipolemoment. Polar solvents and polar aprotic solvents can be present in bothaliphatic and aromatic or cyclic form. In some embodiments, the polarsolvent is acetonitrile.

The organic solvent described herein can have a dielectric constant thatis the same as or close to acetonitrile. The dielectric constant of theorganic solvent can be in the range of about 20-60, about 25-55, about25-50, about 25-45, about 25-40, about 30-50, about 30-45, or about30-40. The dielectric constant of the organic solvent can be greaterthan or equal to about 20, 25, 30, 35, or 40. The dielectric constant ofthe organic solvent can be lower than 30, 40, 45, 50, 55, or 60. Thedielectric constant of the organic solvent can be about 35, 36, 37, 38,or 39.

Dielectric constant may be measured using a test capacitor.Representative polar aprotic solvents having a dielectric constantbetween 30 and 120 may be used. Such solvents may particularly include,but are not limited to, acetonitrile, diethylene glycol,N,N-dimethylacetamide, dimethyl formamide, dimethyl sulfoxide, ethyleneglycol, formamide, hexamethylphosphoramide, glycerin, methanol,N-methyl-2-pyrrolidinone, nitrobenzene, or nitromethane.

The organic solvent described herein can have a polarity index that isthe same as or close to acetonitrile. The polarity index of the organicsolvent can be in the range of about 2-9, 2-8, 2-7, 2-6, 3-9, 3-8, 3-7,3-6, 4-9, 4-8, 4-7, or 4-6. The polarity index of the organic solventcan be greater than, or equal to, about 2, 3, 4, 4.5, 5, 5.5, or 6. Thepolarity index of the organic solvent can be lower than about 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 10. The polarity index of the organicsolvent can be about 5.5, 5.6, 5.7, or 5.8.

The Snyder Polarity Index may be calculated according to the methodsdisclosed in Snyder, L. R., Journal of Chromatography A, 92(2):223-30(1974), which is incorporated by reference herein in its entirety.Representative polar aprotic solvents having a Snyder polarity indexbetween 6.2 and 7.3 may be used. Such solvents may particularly include,but are not limited to, acetonitrile, dimethyl acetamide, dimethylformamide, N-methyl pyrrolidone, N,N-dimethyl sulfoxide, methanol, orformamide.

Relative polarity may be determined according to the methods given inReichardt, C., Solvents and Solvent Effects in Organic Chemistry, 3rded., 2003, which is incorporated herein by reference in its entirety,and especially with respect to its disclosure of polarities and methodsof determining or assessing the same for solvents and solvent molecules.Polar aprotic solvents having a relative polarity between 0.44 and 0.82may be used. Such solvents may particularly include, but are not limitedto, dimethylsulfoxide, acetonitrile, 3-pentanol, 2-pentanol, 2-butanol,Cyclohexanol, 1-octanol, 2-propanol, 1-heptanol, i-butanol, 1-hexanol,1-pentanol, acetyl acetone, ethyl acetoacetate, 1-butanol, benzylalcohol, 1-propanol, 2-aminoethanol, Ethanol, diethylene glycol,methanol, ethylene glycol, glycerin, or formamide.

The Solvent Polarity (E_(T)(30)) may be calculated according to themethods disclosed in Reichardt, C., Molecular Interactions, Volume 3,Ratajczak, H. and Orville, W. J., Eds (1982), which is incorporated byreference herein in its entirety.

Some examples of organic solvents include, but are not limited to,acetonitrile, dimethylformamide (DMF), dimethylsulfoxide (DMSO),acetanilide, N-acetyl pyrrolidone, 4-amino pyridine, benzamide,benzimidazole, 1,2,3-benzotriazole, butadienedioxide, 2,3-butylenecarbonate, γ-butyrolactone, caprolactone (epsilon), chloro maleicanhydride, 2-chlorocyclohexanone, chloroethylene carbonate,chloronitromethane, citraconic anhydride, crotonlactone,5-cyano-2-thiouracil, cyclopropylnitrile, dimethyl sulfate, dimethylsulfone, 1,3-dimethyl-5-tetrazole, 1,5-dimethyl tetrazole,1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,epsilon-caprolactam, ethanesulfonylchloride, ethyl ethyl phosphinate,N-ethyl tetrazole, ethylene carbonate, ethylene trithiocarbonate,ethylene glycol sulfate, ethylene glycol sulfite, furfural,2-furonitrile, 2-imidazole, isatin, isoxazole, malononitrile, 4-methoxybenzonitrile, 1-methoxy-2-nitrobenzene, methyl alpha bromo tetronate,1-methyl imidazole, N-methyl imidazole, 3-methyl isoxazole, N-methylmorpholine-N-oxide, methyl phenyl sulfone, N-methyl pyrrolidinone,methyl sulfolane, methyl-4-toluenesulfonate, 3-nitroaniline,nitrobenzimidazole, 2-nitrofuran, 1-nitroso-2-pyrolidinone,2-nitrothiophene, 2-oxazolidinone, 9,10-phenanthrenequinone, N-phenylsydnone, phthalic anhydride, picolinonitrile (2-cyanopyridine),1,3-propane sultone, β-propiolactone, propylene carbonate,4H-pyran-4-thione, 4H-pyran-4-one (γ-pyrone), pyridazine, 2-pyrrolidone,saccharin, succinonitrile, sulfanilamide, sulfolane,2,2,6,6-tetrachlorocyclohexanone, tetrahydrothiapyran oxide,tetramethylene sulfone (sulfolane), thiazole, 2-thiouracil,3,3,3-trichloro propene, 1, 1,2-trichloro propene, 1,2,3-trichloropropene, trimethylene sulfide-dioxide, or trimethylene sulfite.

Polar aprotic solvents having a solvent polarity between 44 and 60 maybe used. Such solvents may particularly include, but are not limited to,dimethyl sulfoxide, 2-methoxycarbonylphenol, triethyl phosphite,3-pentanol, acetonitrile, nitromethane, cyclohexanol, 2-pentanol,4-methyl-1,3, dioxolan-2-one, propylene carbonate, acrylonitrile,1-phenylethanol, 1-dodecanol, 2-butanol, 2-methylcyclohexanol,2,6,dimethylphenol, 2,6-xylenol, 1-decanol, cyclopentanol, dimethylsulfone, 1-octanoldiethylene glycol mono n-butyl ether, butyl digol,1-heptanol, 3-phenyl-1-propanol, 1,3-dioxolane-2-one, ethylenecarbonate, 1-hexanol, 4-chlorobutyronitrile, 5-methyl-2-isopropylphenol,thymol, 3,5,5-trimethyl-1-hexanol, 3-methyl-1-butanol, isoamyl alcohol,2-methyl-1-propanol, isobutyl alcohol, 2-(tert-butyl)phenol, 1-pentanol,2-phenylethanol, 2-methylpentane-2,4-diol, dipropylene glycol,2-isopropylphenol, 2-n-butoxyethanol, ethylene glycol mono-n-butylether, 1-butanol, 2-hydroxymethyl-tetrahydrofuran, tetrahydrofurfurylalcohol, 2-hydroxymethylfuran, furfuryl alcohol, 1-propanol,2,4-dimethylphenol, 2,4-xylenol, benzyl alcohol, 2-ethoxyphenol,2-ethoxyethanol, 1,5-pentanediol, 1-bromo-2-propanol,2-methyl-5-isopropylphenol, carvacrol, 2-aminoethanol, ethanol,n-methylacetamide, 3-chloropropionitrile, 2-propen-1-ol, allyl alcohol,2-methoxy ethanol, 2-methylphenol, o-cresol, 1,3-butanediol,2-propyn-1-ol, propargyl alcohol, 3-methylphenol, m-cresol, triethyleneglycol, diethylene glycol, n-methylformamide, 1,2-propanediol,1,3-propanediol, 2-chlorophenol, methanol, 1,2-ethanediol, glycol,formamide, 2,2,2-trichloroethanol, 1,2,3-propanetriol, glycerol,2,2,3,3-tetrafluoro-1-propanol, 2,2,2-trifluoroethanol, 4-n-butylphenol,4-methylphenol, or p-cresol.

Polar aprotic solvents having a dielectric constant in the range ofabout 30-115 may be used. Such solvents may particularly include, butare not limited to, dimethyl sulfoxide, 2-methoxycarbonylphenol,triethyl phosphite, 3-pentanol, acetonitrile, nitromethane,cyclohexanol, 2-pentanol, 4-methyl-1,3, dioxolan-2-one, propylenecarbonate, acrylonitrile, 1-phenylethanol, 1-dodecanol, 2-butanol,2-methylcyclohexanol, 2,6,dimethylphenol, 2,6-xylenol, 1-decanol,cyclopentanol, dimethyl sulfone, 1-octanoldiethylene glycol mono n-butylether, butyl digol, 1-heptanol, 3-phenyl-1-propanol,1,3-dioxolane-2-one, ethylene carbonate, 1-hexanol,4-chlorobutyronitrile, 5-methyl-2-isopropylphenol, thymol,3,5,5-trimethyl-1-hexanol, 3-methyl-1-butanol, isoamyl alcohol,2-methyl-1-propanol, isobutyl alcohol, 2-(tert-butyl)phenol, 1-pentanol,2-phenylethanol, 2-methylpentane-2,4-diol, dipropylene glycol,2-isopropylphenol, 2-n-butoxyethanol, ethylene glycol mono-n-butylether, 1-butanol, 2-hydroxymethyl-tetrahydrofuran, tetrahydrofurfurylalcohol, 2-hydroxymethylfuran, furfuryl alcohol, 1-propanol,2,4-dimethylphenol, 2,4-xylenol, benzyl alcohol, 2-ethoxyphenol,2-ethoxyethanol, 1,5-pentanediol, 1-bromo-2-propanol,2-methyl-5-isopropylphenol, carvacrol, 2-aminoethanol, ethanol,n-methylacetamide, 3-chloropropionitrile, 2-propen-1-ol, allyl alcohol,2-methoxyethanol, 2-methylphenol, o-cresol, 1,3-butanediol,2-propyn-1-ol, propargyl alcohol, 3-methylphenol, m-cresol, triethyleneglycol, diethylene glycol, n-methylformamide, 1,2-propanediol,1,3-propanediol, 2-chlorophenol, methanol, 1,2-ethanediol, glycol,formamide, 2,2,2-trichloroethanol, 1,2,3-propanetriol, glycerol,2,2,3,3-tetrafluoro-1-propanol, 2,2,2-trifluoroethanol, 4-n-butylphenol,4-methylphenol, or p-cresol.

Organic solvent addition: In some instances, the disclosed hybridizationbuffer formulations include the addition of an organic solvent. Examplesof suitable solvents include, but are not limited to, acetonitrile,ethanol, DMF, and methanol, or any combination thereof at varyingpercentages (for example >5%). In some instances, the percentage oforganic solvent (by volume) included in the hybridization buffer mayrange from about 1% to about 20%. In some instances, the percentage byvolume of organic solvent may be at least 1%, at least 2%, at least 3%,at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, atleast 9%, at least 10%, at least 15%, or at least 20%. In someinstances, the percentage by volume of organic solvent may be at most20%, at most 15%, at most 10%, at most 9%, at most 8%, at most 7%, atmost 6%, at most 5%, at most 4%, at most 3%, at most 2%, or at most 1%.Any of the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, the percentage by volume of organic solvent may range fromabout 4% to about 15%. The percentage by volume of organic solvent mayhave any value within this range, e.g., about 7.5%.

When the organic solvent comprises a polar aprotic solvent, the amountof the polar aprotic solvent is present in an amount effective todenature a double stranded nucleic acid. In some instances, the amountof the polar aprotic solvent is greater than, or equal to, about 10% byvolume based on the total volume of the formulation. In some instances,the amount of the polar aprotic solvent is about, or more than about,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, orhigher, by volume based on the total volume of the formulation. In someinstances, the amount of the polar aprotic solvent is lower than about15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or higher, byvolume based on the total volume of the formulation. In someembodiments, the amount of the polar aprotic solvent is in the range ofabout 10% to 90% by volume based on the total volume of the formulation.In some instances, the amount of the polar aprotic solvent is in therange of about 25% to 75% by volume based on the total volume of theformulation. In some instances, the amount of the polar aprotic solventis in the range of about 10% to 95%, 10% to 85%, 20% to 90%, 20% to 80%,20% to 75%, or 30% to 60% by volume based on the total volume of theformulation. In some instances, the polar aprotic solvent is formamide.

When the organic solvent comprises a polar aprotic solvent, the amountof the aprotic solvent is present in an amount effective to denature adouble stranded nucleic acid. In some instances, the amount of theaprotic solvent is greater than, or equal to, about 10% by volume basedon the total volume of the formulation. In some instances, the amount ofthe aprotic solvent is about, or more than about, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or higher, by volume basedon the total volume of the formulation. In some instances, the amount ofthe aprotic solvent is lower than about 15%, 20%, 25%, 30%, 35%, 40%,50%, 60%, 70%, 80%, 90%, or higher, by volume based on the total volumeof the formulation. In some instances, the amount of the aprotic solventis in the range of about 10% to 90% by volume based on the total volumeof the formulation. In some instances, the amount of the aprotic solventis in the range of about 25% to 75% by volume based on the total volumeof the formulation. In some instances, the amount of the aprotic solventis in the range of about 10% to 95%, 10% to 85%, 20% to 90%, 20% to 80%,20% to 75%, or 30% to 60% by volume based on the total volume of theformulation.

Addition of molecular crowding/volume exclusion agents: The compositiondescribed herein can include one or more crowding agents that enhancesmolecular crowding. In some instances, the crowding agent is selectedfrom the group consisting of polyethylene glycol (PEG), dextran,hydroxypropyl methyl cellulose (HPMC), hydroxyethyl methyl cellulose(HEMC), hydroxybutyl methyl cellulose, hydroxypropyl cellulose,methycellulose, and hydroxyl methyl cellulose, and combinations thereof.An example crowding agent may comprise one or more of polyethyleneglycol (PEG), dextran, proteins, for example, ovalbumin or hemoglobin,or Ficoll.

A suitable amount of a crowding agent in the composition allows for,enhances, or facilitates molecular crowding. In some instances, theamount of the crowding agent is about, or more than about, 1%, 2%, 3%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, or higher, by volumebased on the total volume of the formulation. In some instances, theamount of the molecular crowding agent is greater than or equal to about5% by volume based on the total volume of the formulation. In someinstances, the amount of the crowding agent is lower than about 3%, 5%,10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, orhigher, by volume based on the total volume of the formulation. In someinstances, the amount of the molecular crowding agent is less than orequal to about 30% by volume based on the total volume of theformulation. In some instances, the amount of the organic solvent is inthe range of about 25% to 75% by volume based on the total volume of theformulation. In some instances, the amount of the organic solvent is inthe range of about 1% to 40%, 1% to 35%, 2% to 50%, 2% to 40%, 2% to35%, 2% to 30%, 2% to 25%, 2% to 20%, 2% to 10%, 5% to 50%, 5% to 40%,5% to 35%, 5% to 30%, 5% to 25%, or 5% to 20% by volume based on thetotal volume of the formulation. In some instances, the amount of themolecular crowding agent is in the range of about 5% to about 20% byvolume based on the total volume of the formulation. In some instances,the amount of the crowding agent is in the range of about 1% to 30% byvolume based on the total volume of the formulation.

One example of the crowding agent in the composition is polyethyleneglycol (PEG). In some instances, the PEG used can have a molecularweight sufficient to enhance or facilitate molecular crowding. In someinstances, the PEG used in the composition has a molecular weight in therange of about 5 k-50 k Da. In some instances, the PEG used in thecomposition has a molecular weight in the range of about 10 k-40 k Da.In some instances, the PEG used in the composition has a molecularweight in the range of about 10 k-30 k Da. In some instances, the PEGused in the composition has a molecular weight in the range of about 20k Da.

In some instances, the disclosed hybridization buffer formulations mayinclude the addition of a molecular crowding or volume exclusion agent.Molecular crowding or volume exclusion agents are, for example,macromolecules (e.g., proteins) which, when added to a solution in highconcentrations, may alter the properties of other molecules in solutionby reducing the volume of solvent available to the other molecules. Insome instances, the percentage by volume of the molecular crowding orvolume exclusion agent included in the hybridization buffer formulationmay range from about 1% to about 50%. In some instances, the percentageby volume of the molecular crowding or volume exclusion agent may be atleast 1%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, or atleast 50%. In some instances, the percentage by volume of the molecularcrowding or volume exclusion agent may be at most 50%, at most 45%, atmost 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most15%, at most 10%, at most 5%, or at most 1%. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, the percentage byvolume of molecular crowding or volume exclusion agent may range fromabout 5% to about 35%. The percentage by volume of molecular crowding orvolume exclusion agent may have any value within this range, e.g., about12.5%.

PH buffer system: The compositions described herein include a pH buffersystem that maintains the pH of the compositions in a range suitable forhybridization process. The pH buffer system can include one or morebuffering agents selected from the group consisting of Tris, HEPES,TAPS, Tricine, Bicine, Bis-Tris, NaOH, KOH, TES, EPPS, MES, and MOPS.The pH buffer system can further include a solvent. An example pH buffersystem includes MOPS, IVIES, TAPS, phosphate buffer combined withmethanol, acetonitrile, ethanol, isopropanol, butanol, t-butyl alcohol,DMF, DMSO, or any combination therein.

The amount of the pH buffer system is effective to maintain the pH ofthe formulation in a range suitable for hybridization. In someinstances, the pH may be at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, or at least 10. In some instances,the pH may be at most 10, at most 9, at most 8, at most 7, at most 6, atmost 5, at most 4, or at most 3. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, the pH of the hybridizationbuffer may range from about 4 to about 8. The pH of the hybridizationbuffer may have any value within this range, e.g., about pH 7.8. In somecases, the pH range is about 3 to about 10. In some instances, thedisclosed hybridization buffer formulations may include adjustment of pHover the range of about pH 3 to pH 10, with a narrower buffer range of5-9.

Additives that impact DNA melting temperatures: The compositionsdescribed herein can include one or more additives to allow for bettercontrol of the melting temperature of the nucleic acid and enhance thestringency control of the hybridization reaction. Hybridizationreactions are usually carried out under stringent conditions in order toachieve hybridization specificity. In some cases, the additive forcontrolling melting temperature of nucleic acid is formamide.

The amount of the additive for controlling melting temperature ofnucleic acid can vary depending on other agents used in thecompositions. In some instances, the amount of the additive forcontrolling melting temperature of the nucleic acid is about, or morethan about, 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%,or higher by volume based on the total volume of the formulation. Insome instances, the amount of the additive for controlling the meltingtemperature of the nucleic acid is greater than or equal to about 2% byvolume based on the total volume of the formulation. In some instances,the amount of the additive for controlling the melting temperature ofthe nucleic acid is greater than or equal to about 5% by volume based onthe total volume of the formulation. In some instances, the amount ofthe additive for controlling the melting temperature of the nucleic acidis lower than about 3%, 5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%,50%, 60%, 70%, 80%, 90%, or higher, by volume based on the total volumeof the formulation. In some instances, the amount of the additive forcontrolling the melting temperature of the nucleic acid is in the rangeof about 1% to 40%, 1% to 35%, 2% to 50%, 2% to 40%, 2% to 35%, 2% to30%, 2% to 25%, 2% to 20%, 2% to 10%, 5% to 50%, 5% to 40%, 5% to 35%,5% to 30%, 5% to 25%, or 5% to 20% by volume based on the total volumeof the formulation. In some instances, the amount of the additive forcontrolling melting temperature of the nucleic acid is in the range ofabout 2% to 20% by volume based on the total volume of the formulation.In some instances, the amount of the additive for controlling meltingtemperature of the nucleic acid is in the range of about 5% to 10% byvolume based on the total volume of the formulation.

In some instances, the disclosed hybridization buffer formulations mayinclude the addition of an additive that alters nucleic acid duplexmelting temperature. Examples of suitable additives that may be used toalter nucleic acid melting temperature include, but are not limited to,formamide. In some instances, the percentage by volume of a meltingtemperature additive included in the hybridization buffer formulationmay range from about 1% to about 50%. In some instances, the percentageby volume of a melting temperature additive may be at least 1%, at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, or at least 50%. In someinstances, the percentage by volume of a melting temperature additivemay be at most 50%, at most 45%, at most 40%, at most 35%, at most 30%,at most 25%, at most 20%, at most 15%, at most 10%, at most 5%, or atmost 1%. Any of the lower and upper values described in this paragraphmay be combined to form a range included within the present disclosure,for example, the percentage by volume of a melting temperature additivemay range from about 10% to about 25%. The percentage by volume of amelting temperature additive may have any value within this range, e.g.,about 22.5%.

Additives that impact DNA hydration: In some instances, the disclosedhybridization buffer formulations include the addition of an additivethat impacts nucleic acid hydration. Examples include, but are notlimited to, betaine, urea, glycine betaine, or any combination thereof.In some instances, the percentage by volume of a hydration additiveincluded in the hybridization buffer formulation ranges from about 1% toabout 50%. In some instances, the percentage by volume of a hydrationadditive is at least 1%, at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, or at least 50%. In some instances, the percentage by volumeof a hydration additive is at most 50%, at most 45%, at most 40%, atmost 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most10%, at most 5%, or at most 1%. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure. For example, the percentage by volume ofa hydration additive may range from about 1% to about 30%. Thepercentage by volume of a melting temperature additive may have anyvalue within this range, e.g., about 6.5%.

Systems

Provided herein are systems comprising the hybridization compositionsdescribed herein and a low non-specific binding surface. The systemsdescribed herein, in some instances, comprise a flow cell device. Thesystems described herein further comprise, in some instances, an imagingsystem (e.g., a camera and an inverted fluorescent microscope). Systemsmay further comprise one or more computer control systems to performcomputer-implemented methods of nucleic acid analysis.

Low non-specific binding surface: Disclosed herein is a low non-specificbinding surface that enables improved nucleic acid hybridization andamplification performance. The disclosed surface may comprise one ormore layers of covalently or non-covalently attached low-bindingchemical modification layers, e.g., silane layers or polymer films, andone or more covalently or non-covalently attached primer sequences thatmay be used for tethering single-stranded template oligonucleotides tothe surface. In some instances, the formulation of the surface, e.g.,the chemical composition of the one or more layers, the couplingchemistry used to cross-link the one or more layers to the surface, or,to each other or a combination thereof, and the total number of layers,may be varied such that non-specific binding of proteins, nucleic acidmolecules, and other hybridization and amplification reaction componentsto the surface is minimized or reduced relative to a comparablemonolayer. The formulation of the surface may be varied such thatnon-specific hybridization on the surface is minimized or reducedrelative to a comparable monolayer. The formulation of the surface maybe varied such that non-specific amplification on the surface isminimized or reduced relative to a comparable monolayer. The formulationof the surface may be varied such that specific amplification rates, or,yields, or a combination thereof on the surface are maximized.Amplification levels suitable for detection are achieved in no more than2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 30 amplification cycles in some casesdisclosed herein.

Non-limiting examples of low non-specific binding surfaces are providedin co-pending U.S. patent application Ser. No. 16/739,007, which ishereby incorporated by reference in its entirety. The terms, “lownon-specific binding surface” and “low binding surface” are usedinterchangeably to refer to hydrophilic surfaces that exhibit a lowamount of non-specific binding to proteins or nucleic acids, as comparedwith a surface that is not hydrophilic. In some instances, the lownon-specific binding surface is passivated, meaning it is coated with asubstrate that is hydrophilic.

Examples of materials from which the substrate or support structure maybe fabricated include, but are not limited to, glass, fused-silica,silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene(MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene(PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefinpolymers (COP), cyclic olefin copolymers (COC), polyethyleneterephthalate (PET)), or any combination thereof. Various compositionsof both glass and plastic substrates are contemplated.

The substrate or support structure may be rendered in any of a varietyof geometries and dimensions, and may comprise any of a variety ofmaterials. For example, in some instances, the substrate or supportstructure is locally planar (e.g., comprising a microscope slide or thesurface of a microscope slide). Globally, the substrate or supportstructure may be cylindrical (e.g., comprising a capillary or theinterior surface of a capillary), spherical (e.g., comprising the outersurface of a non-porous bead), or irregular (e.g., comprising the outersurface of an irregularly-shaped, non-porous bead or particle). In someinstances, the surface of the substrate or support structure used fornucleic acid hybridization and amplification may be a solid, non-poroussurface. In some instances, the surface of the substrate or supportstructure used for nucleic acid hybridization and amplification may beporous, such that the coatings described herein penetrate the poroussurface, and nucleic acid hybridization and amplification reactionsperformed thereon may occur within the pores.

The substrate or support structure that comprises the one or morechemically-modified layers, e.g., layers of a low non-specific bindingpolymer, may be independent or integrated into another structure orassembly. For example, in some instances, the substrate or supportstructure comprises one or more surfaces within an integrated orassembled microfluidic flow cell. The substrate or support structure maycomprise one or more surfaces within a microplate format, e.g., thebottom surface of the wells in a microplate. As noted above, in someinstances, the substrate or support structure comprises the interiorsurface (such as the lumen surface) of a capillary. In another example,the substrate or support structure comprises the interior surface (suchas the lumen surface) of a capillary etched into a planar chip.

The chemical modification layers may be applied uniformly across thesurface of the substrate or support structure. In another example, thesurface of the substrate or support structure may be non-uniformlydistributed or patterned, such that the chemical modification layers areconfined to one or more discrete regions of the substrate. For example,the substrate surface may be patterned using photolithographictechniques to create an ordered array or random pattern ofchemically-modified regions on the surface. The substrate surface may bepatterned using contact printing, or, ink-jet printing techniques, or acombination thereof. In some instances, an ordered array or randompatter of chemically-modified discrete regions may comprise at least 1,5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or10,000 or more discrete regions, or any intermediate number spanned bythe range herein.

In order to achieve low non-specific binding surfaces (also referred toherein as “low binding” or “passivated” surfaces), hydrophilic polymersmay be non-specifically adsorbed or covalently grafted to the substrateor support surface. For example, passivation can be performed utilizingpoly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) orpolyoxyethylene), poly(vinyl alcohol) (PVA), poly(vinyl pyridine),poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA),poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA),poly-lysine, poly-glucoside, streptavidin, dextran, or other hydrophilicpolymers with different molecular weights and end groups that are linkedto a surface using, for example, silane chemistry. The end groups distalfrom the surface can include, but are not limited to, biotin, methoxyether, carboxylate, amine, NHS ester, maleimide, and bis-silane. In someinstances, two or more layers of a hydrophilic polymer, e.g., a linearpolymer, branched polymer, or multi-branched polymer, may be depositedon the surface. In some instances, two or more layers may be covalentlycoupled to each other or internally cross-linked to improve thestability of the resulting surface. In some instances, oligonucleotideprimers with different base sequences and base modifications (or otherbiomolecules, e.g., enzymes or antibodies) may be tethered to theresulting surface layer at various surface densities. In some instances,for example, both surface functional group density and oligonucleotideconcentration may be varied to target a certain primer density range.Additionally, primer density can be controlled by diluting theoligonucleotides with other molecules that carry the same functionalgroup. For example, amine-labeled oligonucleotides can be diluted withamine-labeled polyethylene glycol in a reaction with an NETS-estercoated surface to reduce the final primer density. Primers withdifferent lengths of linker between the hybridization region and thesurface attachment functional group can also be applied to controlsurface density. Examples of suitable linkers include poly-T (SEQ IDNO: 1) and poly-A (SEQ ID NO: 2) strands at the 5′ end of the primer(e.g., 0 to 20 bases), PEG linkers (e.g., 3 to 20 monomer units), andcarbon-chains (e.g., C6, C12, C18, etc.). To measure the primer density,fluorescently-labeled primers may be tethered to the surface and afluorescence reading corresponding to the fluorescently-labeled primersmay then be compared with that for a dye solution of knownconcentration.

As a result of the surface passivation techniques disclosed herein,proteins, nucleic acids, and other biomolecules do not “stick” tosubstrates, that is, they exhibit low non-specific binding (non-specificbinding). Examples are shown below using standard monolayer surfacepreparations with varying glass preparation conditions. Hydrophilicsurfaces that have been passivated to achieve ultra-low non-specificbinding for proteins and nucleic acids require novel reaction conditionsto improve primer deposition reaction efficiencies and hybridizationperformance, and to induce effective amplification. All of theseprocesses require oligonucleotide attachment and subsequent proteinbinding and delivery to a low binding surface. As described below, thecombination of a new primer surface conjugation formulation (Cy3oligonucleotide graft titration) and resulting ultra-low non-specificbackground (non-specific binding functional tests performed using redand green fluorescent dyes) yielded results that demonstrate theviability of the disclosed approaches. Some surfaces disclosed hereinexhibit a ratio of specific (e.g., hybridization to a tethered primer orprobe) to non-specific binding (e.g., B_(inter)) of a fluorophore suchas Cy3 of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1,40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediatevalue spanned by the range herein. Some surfaces disclosed hereinexhibit a ratio of specific to non-specific fluorescence signal (e.g.,for specifically-hybridized to non-specifically bound labeledoligonucleotides, or for specifically-amplified tonon-specifically-bound (B_(inter)) or non-specifically amplified(B_(intra)) labeled oligonucleotides or a combination thereof (B_(inter)B_(intra))) for a fluorophore such as Cy3 of at least 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greaterthan 100:1, or any intermediate value spanned by the range herein.

In order to scale primer surface density and add additionaldimensionality to hydrophilic or amphoteric surfaces, substratescomprising multi-layer coatings of PEG and other hydrophilic polymershave been developed. By using hydrophilic and amphoteric surfacelayering approaches that include, but are not limited to, thepolymer/co-polymer materials described below, it is possible to increaseprimer loading density on the surface significantly. Traditional PEGcoating approaches use monolayer primer deposition, which has beengenerally reported as successful for single molecule applications, butdoes not yield high copy numbers for nucleic acid amplificationapplications. As described herein, “layering” can be accomplished usingtraditional crosslinking approaches with any compatible polymer ormonomer subunits, such that a surface comprising two or more highlycrosslinked layers can be built sequentially. Examples of suitablepolymers include, but are not limited to, streptavidin, poly acrylamide,polyester, dextran, poly-lysine, and copolymers of poly-lysine and PEG.In some instances, the different layers may be attached to each otherthrough any of a variety of conjugation reactions including, but notlimited to, biotin-streptavidin binding, azide-alkyne click reaction,amine-NETS ester reaction, thiol-maleimide reaction, and ionicinteractions between positively charged polymers and negatively chargedpolymers. In some instances, high primer density materials may beconstructed in solution and subsequently layered onto the surface inmultiple operations.

The attachment chemistry used to graft a first chemically-modified layerto a support surface will generally be dependent on both the materialfrom which the support is fabricated and the chemical nature of thelayer. In some instances, the first layer may be covalently attached tothe support surface. In some instances, the first layer may benon-covalently attached, e.g., adsorbed to the surface throughnon-covalent interactions such as electrostatic interactions, hydrogenbonding, or van der Waals interactions between the surface and themolecular components of the first layer. In either case, the substratesurface may be treated prior to attachment or deposition of the firstlayer. Any of a variety of surface preparation techniques may be used toclean or treat the support surface. For example, glass or siliconsurfaces may be acid-washed using a Piranha solution (a mixture ofsulfuric acid (H₂SO₄) and hydrogen peroxide (H₂O₂)), or, cleaned usingan oxygen plasma treatment method, or a combination thereof.

Silane chemistries constitute one non-limiting approach for covalentlymodifying the silanol groups on glass or silicon surfaces to attach morereactive functional groups (e.g., amines or carboxyl groups), which maythen be used in coupling linker molecules (e.g., linear hydrocarbonmolecules of various lengths, such as C6, C12, C18 hydrocarbons, orlinear polyethylene glycol (PEG) molecules) or layer molecules (e.g.,branched PEG molecules or other polymers) to the surface. Examples ofsuitable silanes that may be used in creating any of the disclosed lowbinding support surfaces include, but are not limited to,(3-Aminopropyl) trimethoxysilane (APTMS), (3-Aminopropyl)triethoxysilane (APTES), any of a variety of PEG-silanes (e.g.,comprising molecular weights of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEGsilane (e.g., comprising a free amino functional group), maleimide-PEGsilane, biotin-PEG silane, and the like.

Any of a variety of molecules including, but not limited to, aminoacids, peptides, nucleotides, oligonucleotides, other monomers orpolymers, or combinations thereof may be used in creating the one ormore chemically-modified layers on the support surface, where the choiceof components used may be varied to alter one or more properties of thesupport surface, e.g., the surface density of functional groups, or,tethered oligonucleotide primers, or a combination thereof; thehydrophilicity/hydrophobicity of the support surface, or the threethree-dimensional nature (e.g., “thickness”) of the support surface.Examples of polymers that may be used to create one or more layers oflow non-specific binding material in any of the disclosed supportsurfaces include, but are not limited to, polyethylene glycol (PEG) ofvarious molecular weights and branching structures, streptavidin,polyacrylamide, polyester, dextran, poly-lysine, and poly-lysinecopolymers, or any combination thereof. Examples of conjugationchemistries that may be used to graft one or more layers of material(e.g., polymer layers) to the support surface, or, to cross-link thelayers to each other, or a combination thereof include, but are notlimited to, biotin-streptavidin interactions (or variations thereof),his tag—Ni/NTA conjugation chemistries, methoxy ether conjugationchemistries, carboxylate conjugation chemistries, amine conjugationchemistries, NHS esters, maleimides, thiol, epoxy, azide, hydrazide,alkyne, isocyanate, and silane.

One or more layers of a multi-layered surface may comprise a branchedpolymer or may be linear. Examples of suitable branched polymersinclude, but are not limited to, branched PEG, branched poly(vinylalcohol) (branched PVA), branched poly(vinyl pyridine), branchedpoly(vinyl pyrrolidone) (branched PVP), branched), poly(acrylic acid)(branched PAA), branched polyacrylamide, branchedpoly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methylmethacrylate) (branched PMA), branched poly(2-hydroxylethylmethacrylate) (branched PHEMA), branched poly(oligo(ethylene glycol)methyl ether methacrylate) (branched POEGMA), branched polyglutamic acid(branched PGA), branched poly-lysine, branched poly-glucoside, anddextran.

In some instances, the branched polymers used to create one or morelayers of any of the multi-layered surfaces disclosed herein maycomprise at least 4 branches, at least 5 branches, at least 6 branches,at least 7 branches, at least 8 branches, at least 9 branches, at least10 branches, at least 12 branches, at least 14 branches, at least 16branches, at least 18 branches, at least 20 branches, at least 22branches, at least 24 branches, at least 26 branches, at least 28branches, at least 30 branches, at least 32 branches, at least 34branches, at least 36 branches, at least 38 branches, or at least 40branches. Molecules often exhibit a ‘power of 2’ number of branches,such as 2, 4, 8, 16, 32, 64, or 128 branches.

PEG multilayers comprising PEG (8,16,8) on PEG-amine-APTES exposed totwo layers of 7 uM primer pre-loading exhibited a concentration of2,000,000 to 10,000,000 on the surface. Similar concentrations wereobserved for 3-layer multi-arm PEG (8,16,8) and (8,64,8) onPEG-amine-APTES exposed to 8 uM primer, and 3-layer multi-arm PEG(8,8,8) using star-shaped PEG-amine to replace dumbbell-shaped 16 merand 64 mer. PEG multilayers having comparable first, second and thirdPEG levels are also contemplated.

Linear, branched, or multi-branched polymers used to create one or morelayers of any of the multi-layered surfaces disclosed herein may have amolecular weight of at least 500, at least 1,000, at least 2,000, atleast 3,000, at least 4,000, at least 5,000, at least 10,000, at least15,000, at least 20,000, at least 25,000, at least 30,000, at least35,000, at least 40,000, at least 45,000, or at least 50,000 daltons.

In some instances, e.g., wherein at least one layer of a multi-layeredsurface comprises a branched polymer, the number of covalent bondsbetween a branched polymer molecule of the layer being deposited andmolecules of the underlying layer may range from about one covalentlinkage per molecule to about 32 covalent linkages per molecule. In someinstances, the number of covalent bonds between a branched polymermolecule of the new layer and molecules of the underlying layer may beat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 12, at least14, at least 16, at least 18, at least 20, at least 22, at least 24, atleast 26, at least 28, at least 30, or at least 32 or more than 32covalent linkages per molecule.

Any reactive functional groups that remain following the coupling of amaterial layer to the support surface may optionally be blocked bycoupling a small, inert molecule using a high yield coupling chemistry.For example, in the case that amine coupling chemistry is used to attacha new material layer to the underlying one, any residual amine groupsmay subsequently be acetylated or deactivated by coupling with a smallamino acid such as glycine.

The number of layers of low non-specific binding material, e.g., ahydrophilic polymer material, deposited on the surface of the disclosedlow binding supports may range from 1 to about 10. In some instances,the number of layers is at least 1, at least 2, at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, or at least10. In some instances, the number of layers may be at most 10, at most9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, atmost 2, or at most 1. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the number of layersmay range from about 2 to about 4. In some instances, all of the layersmay comprise the same material. In some instances, each layer maycomprise a different material. In some instances, the plurality oflayers may comprise a plurality of materials. In some instances, atleast one layer may comprise a branched polymer. In some instances, allof the layers may comprise a branched polymer.

One or more layers of low non-specific binding material may, in somecases, be deposited on, or, conjugated to the substrate surface, or acombination thereof, using a polar protic solvent, a polar aproticsolvent, a nonpolar solvent, or any combination thereof. In someinstances, the solvent used for layer deposition, or, coupling, or acombination thereof may comprise an alcohol (e.g., methanol, ethanol,propanol, etc.), another organic solvent (e.g., acetonitrile, dimethylsulfoxide (DMSO), dimethyl formamide (DMF), etc.), water, an aqueousbuffer solution (e.g., phosphate buffer, phosphate buffered saline,3-(N-morpholino)propanesulfonic acid (MOPS), etc.), or any combinationthereof. In some instances, an organic component of the solvent mixtureused may comprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of thetotal, or any percentage spanned or adjacent to the range herein, withthe balance made up of water or an aqueous buffer solution. In someinstances, an aqueous component of the solvent mixture used may compriseat least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, or anypercentage spanned or adjacent to the range herein, with the balancemade up of an organic solvent. The pH of the solvent mixture used may beless than or equal to about 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,or any value spanned or adjacent to the range described herein. The pHof the solvent mixture may be greater than or equal to about 10.

In some instances, one or more layers of low non-specific bindingmaterial may be deposited on, or, conjugated to the substrate surface,or a combination thereof, using a mixture of organic solvents, whereinthe dielectric constant of at least once component is less than 40 andconstitutes at least 50% of the total mixture by volume. In someinstances, the dielectric constant of the at least one component may beless than 10, less than 20, less than 30, or less than 40. In someinstances, the at least one component constitutes at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least80% of the total mixture by volume.

As noted, the low non-specific binding supports of the presentdisclosure exhibit reduced non-specific binding of proteins, nucleicacids, and other components of the hybridization, or, amplificationformulation, or a combination thereof, used for solid-phase nucleic acidamplification. The degree of non-specific binding exhibited by a givensupport surface may be assessed either qualitatively or quantitatively.For example, in some instances, exposure of the surface to fluorescentdyes (e.g., Cy3, Cy5, etc.), fluorescently-labeled nucleotides,fluorescently-labeled oligonucleotides, or, fluorescently-labeledproteins (e.g., polymerases), or a combination thereof, under astandardized set of conditions, followed by a specified rinse protocoland fluorescence imaging may be used as a qualitative tool forcomparison of non-specific binding on supports comprising differentsurface formulations. In some instances, exposure of the surface tofluorescent dyes, fluorescently-labeled nucleotides,fluorescently-labeled oligonucleotides, or, fluorescently-labeledproteins (e.g., polymerases), or a combination thereof, under astandardized set of conditions, followed by a specified rinse protocoland fluorescence imaging may be used as a quantitative tool forcomparison of non-specific binding on supports comprising differentsurface formulations—provided that care has been taken to ensure thatthe fluorescence imaging is performed under conditions wherefluorescence signal is linearly related (or related in a predictablemanner) to the number of fluorophores on the support surface (e.g.,under conditions where signal saturation, or, self-quenching of thefluorophore, or a combination thereof, is not an issue) and suitablecalibration standards are used. In some instances, other techniques, forexample, radioisotope labeling and counting methods may be used forquantitative assessment of the degree to which non-specific binding isexhibited by the different support surface formulations of the presentdisclosure.

Some surfaces disclosed herein exhibit a ratio of specific tonon-specific binding of a fluorophore such as Cy3 of at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 50, 75, 100, or greater than 100, or any intermediate value spannedby the range herein. Some surfaces disclosed herein exhibit a ratio ofspecific to non-specific fluorescence of a fluorophore such as Cy3 of atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or anyintermediate value spanned by the range herein.

As noted, in some instances, the degree of non-specific bindingexhibited by the disclosed low-binding supports may be assessed using astandardized protocol for contacting the surface with a labeled protein(e.g., bovine serum albumin (BSA), streptavidin, a DNA polymerase, areverse transcriptase, a helicase, a single-stranded binding protein(SSB), etc., or any combination thereof), a labeled nucleotide, alabeled oligonucleotide, etc., under a standardized set of incubationand rinse conditions, followed by detection of the amount of labelremaining on the surface and comparison of the signal resultingtherefrom to an appropriate calibration standard. In some instances, thelabel may comprise a fluorescent label. In some instances, the label maycomprise a radioisotope. In some instances, the label may comprise anyother detectable label. In some instances, the degree of non-specificbinding exhibited by a given support surface formulation may thus beassessed in terms of the number of non-specifically bound proteinmolecules (or other molecules) per unit area. In some instances, thelow-binding supports of the present disclosure may exhibit non-specificprotein binding (or non-specific binding of other specified molecules,e.g., Cy3 dye) of less than or equal to about 0.001 molecule per μm²,less than or equal to about 0.01 molecule per μm², less than or equal toabout 0.1 molecule per μm², less than or equal to about 0.25 moleculeper μm², less than or equal to about 0.5 molecule per μm², less than orequal to about 1 molecule per μm², less than or equal to about 10molecules per μm², less than or equal to about 100 molecules per μm², orless than or equal to about 1,000 molecules per μm². A given supportsurface of the present disclosure may exhibit non-specific bindingfalling anywhere within this range, for example, of less than 86molecules per μm².

In some instances, the surfaces disclosed herein exhibit a ratio ofspecific to non-specific binding of a fluorophore such as Cy3 of atleast or equal to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or any intermediatevalue spanned by the range herein. In some instances, the surfacesdisclosed herein exhibit a ratio of specific to non-specific binding offluorophore such as Cy3 of greater than or equal to about 100. In someinstances, the surfaces disclosed herein exhibit a ratio of specific tonon-specific fluorescence signals for a fluorophore such as Cy3 of atleast or equal to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or any intermediatevalue spanned by the range herein. In some instances, the surfacesdisclosed herein exhibit a ratio of specific to non-specificfluorescence signals for a fluorophore such as Cy3 of greater than orequal to about 100.

The low-background surfaces consistent with the disclosure herein mayexhibit specific dye attachment (e.g., Cy3 attachment) to non-specificdye adsorption (e.g., Cy3 dye adsorption) ratios of at least 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50specific dye molecules attached per molecule non-specifically adsorbed.Similarly, when subjected to an excitation energy, low-backgroundsurfaces consistent with the disclosure herein to which fluorophores,e.g., Cy3, have been attached may exhibit ratios of specificfluorescence signal (e.g., arising from Cy3-labeled oligonucleotidesattached to the surface) to non-specific adsorbed dye fluorescencesignals of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1,30:1, 40:1, 50:1, or more than 50:1.

In some instances, the degree of hydrophilicity (or “wettability” withaqueous solutions) of the disclosed support surfaces may be assessed,for example, through the measurement of water contact angles in which asmall droplet of water is placed on the surface and its angle of contactwith the surface is measured using, e.g., an optical tensiometer. Insome instances, a static contact angle may be determined. In someinstances, an advancing or receding contact angle may be determined. Insome instances, the water contact angle for the hydrophilic, low-bindingsupport surfaced disclosed herein may range from about 0 degrees toabout 30 degrees. In some instances, the water contact angle for thehydrophilic, low-binding support surfaced disclosed herein may be nomore than 50 degrees, 45 degrees, 40 degrees, 30 degrees, 25 degrees, 20degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree. In many cases thecontact angle is no more than 40 degrees. A given hydrophilic,low-binding support surface of the present disclosure may exhibit awater contact angle having a value of anywhere within this range.

In some instances, the hydrophilic surfaces disclosed herein facilitatereduced wash times for bioassays, often due to reduced non-specificbinding of biomolecules to the low-binding surfaces. In some instances,adequate washes may be performed in less than or equal to about 60, 50,40, 30, 20, 15, 10, or less than 10 seconds. For example, in someinstances adequate washes may be performed in less than 30 seconds.

Some low-binding surfaces of the present disclosure exhibit significantimprovement in stability or durability to prolonged exposure to solventsand elevated temperatures, or to repeated cycles of solvent exposure orchanges in temperature. For example, in some instances, the stability ofthe disclosed surfaces may be tested by fluorescently labeling afunctional group on the surface, or a tethered biomolecule (e.g., anoligonucleotide primer) on the surface, and monitoring fluorescencesignal before, during, and after prolonged exposure to solvents andelevated temperatures, or to repeated cycles of solvent exposure orchanges in temperature. In some instances, the degree of change in thefluorescence used to assess the quality of the surface may be less thanor equal to about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% over a timeperiod of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45hours, 50 hours, or 100 hours of exposure to solvents, or, elevatedtemperatures, or a combination thereof (or any combination of thesepercentages as measured over these time periods). In some instances, thedegree of change in the fluorescence used to assess the quality of thesurface may be less than or equal to about 1%, 2%, 3%, 4%, 5%, 10%, 15%,20%, or 25% over 5 cycles, 10 cycles, 20 cycles, 30 cycles, 40 cycles,50 cycles, 60 cycles, 70 cycles, 80 cycles, 90 cycles, 100 cycles, 200cycles, 300 cycles, 400 cycles, 500 cycles, 600 cycles, 700 cycles, 800cycles, 900 cycles, or 1,000 cycles of repeated exposure to solventchanges, or changes in temperature, or a combination thereof (or anycombination of these percentages as measured over this range of cycles).

In some instances, the surfaces disclosed herein may exhibit a highratio of specific signal to non-specific signal or other background. Forexample, when used for nucleic acid amplification, some surfaces mayexhibit an amplification signal that is at least 4-, 5-, 6-, 7-, 8-, 9-,10-, 15-, 20-, 30-, 40-, 50-, 75-, 100-, or greater than 100-foldgreater than a signal of an adjacent unpopulated region of the surface.In some instances, the surfaces exhibit an amplification signal that isat least 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 30-, 40-, 50-, 75-,100-, or greater than 100-fold greater than a signal of an adjacentamplified nucleic acid population region of the surface.

Fluorescence excitation energies vary among particular fluorophores andprotocols, and may range in excitation wavelength, consistent withfluorophore selection or other parameters of use of a surface disclosedherein. In some instances, the wavelength is less than or equal to about400 nanometers (nm). In some instances, the wavelength is more than orequal to about 800 nm. In some instances, the wavelength is between 400nm and 800 nm.

Accordingly, low background surfaces as disclosed herein exhibit lowbackground fluorescence signals or high contrast to noise (CNR) ratios.For example, in some instances, the background fluorescence of thesurface at a location that is spatially distinct or removed from alabeled feature on the surface (e.g., a labeled spot, cluster, discreteregion, sub-section, or subset of the surface) comprising a hybridizedcluster of nucleic acid molecules, or a clonally-amplified cluster ofnucleic acid molecules produced by 20 cycles of nucleic acidamplification via thermocycling, may be no more than 20×, 10×, 5×, 2×,1×, 0.5×, 0.1×, or less than 0.1× greater than the backgroundfluorescence measured at that same location prior to performing saidhybridization or said 20 cycles of nucleic acid amplification.

In some instances, fluorescence images of the disclosed low backgroundsurfaces when used in nucleic acid hybridization or amplificationapplications to create clusters of hybridized or clonally-amplifiednucleic acid molecules (e.g., that have been directly or indirectlylabeled with a fluorophore) exhibit contrast-to-noise ratios (CNRs) ofat least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250, or greater than250.

The surface that comprises the one or more chemically-modified layers,e.g., layers of a low non-specific binding polymer, may be independentor integrated into another structure or assembly. The chemicalmodification layers may be applied uniformly across the surface.Alternately, the surface may be patterned, such that the chemicalmodification layers are confined to one or more discrete regions of thesubstrate. For example, the surface may be patterned usingphotolithographic techniques to create an ordered array or randompattern of chemically-modified regions on the surface. The substratesurface may be patterned using, e.g., contact printing, or, ink-jetprinting techniques, or a combination thereof. In some instances, anordered array or random patter of chemically-modified regions maycomprise at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000,7000, 8000, 9000, or 10,000 or more discrete regions.

In order to achieve low non-specific binding surfaces (also referred toherein as “low binding” or “passivated” surfaces), hydrophilic polymersmay be non-specifically adsorbed or covalently grafted to the surface.For example, passivation can be performed utilizing poly(ethyleneglycol) (PEG, also known as polyethylene oxide (PEO) or polyoxyethylene)or other hydrophilic polymers with different molecular weights and endgroups that are linked to a surface using, for example, silanechemistry. The end groups distal from the surface can include, but arenot limited to, biotin, methoxy ether, carboxylate, amine, NHS ester,maleimide, and bis-silane. In some instances, two or more layers of ahydrophilic polymer, e.g., a linear polymer, branched polymer, ormulti-branched polymer, may be deposited on the surface. In someinstances, two or more layers may be covalently coupled to each other orinternally cross-linked to improve the stability of the resultingsurface. In some instances, oligonucleotide primers with different basesequences and base modifications (or other biomolecules, e.g., enzymesor antibodies) may be tethered to the resulting surface layer at varioussurface densities. In some instances, for example, both surfacefunctional group density and oligonucleotide concentration may be variedto target a certain primer density range. Additionally, primer densitycan be controlled by diluting oligonucleotides with other molecules thatcarry the same functional group. For example, amine-labeledoligonucleotides can be diluted with amine-labeled polyethylene glycolin a reaction with an NETS-ester coated surface to reduce the finalprimer density. Primers with different lengths of linker between thehybridization region and the surface attachment functional group canalso be applied to control surface density. Examples of suitable linkersinclude poly-T (SEQ ID NO: 1) and poly-A (SEQ ID NO: 2) strands at the5′ end of the primer (e.g., 0 to 20 bases), PEG linkers (e.g., 3 to 20monomer units), and carbon-chain (e.g., C6, C12, C18, etc.). To measurethe primer density, fluorescently-labeled primers may be tethered to thesurface and a fluorescence reading corresponding to the primers may thenbe compared with that for a dye solution of known concentration.

As noted, the low non-specific binding surfaces described herein exhibitreduced non-specific binding of nucleic acids, and other components ofthe hybridization, or, amplification formulation, or a combinationthereof used for solid-phase nucleic acid amplification. The degree ofnon-specific binding exhibited by a given surface may be assessed eitherqualitatively or quantitatively. For example, in some instances,exposure of the surface to fluorescent dyes (e.g., Cy3, Cy5, etc.),fluorescently-labeled nucleotides, fluorescently-labeledoligonucleotides, or, fluorescently-labeled proteins (e.g.,polymerases), or a combination thereof under a standardized set ofconditions, followed by a specified rinse protocol and fluorescenceimaging may be used as a qualitative tool for comparison of non-specificbinding surfaces comprising different surface formulations. In someinstances, exposure of the surface to fluorescent dyes,fluorescently-labeled nucleotides, fluorescently-labeledoligonucleotides, or, fluorescently-labeled proteins (e.g.,polymerases), or combination thereof under a standardized set ofconditions, followed by a specified rinse protocol and fluorescenceimaging may be used as a quantitative tool for comparison ofnon-specific binding on surfaces comprising different surfaceformulations—provided that care has been taken to ensure that thefluorescence imaging is performed under conditions where fluorescencesignal is linearly related (or related in a predictable manner) to thenumber of fluorophores on the surface (e.g., under conditions wheresignal saturation, or, self-quenching of the fluorophore, or acombination thereof is not an issue) and suitable calibration standardsare used. In some instances, other techniques, for example, radioisotopelabeling and counting methods may be used for quantitative assessment ofthe degree to which non-specific binding is exhibited by the differentsurface formulations of the present disclosure.

As noted, in some instances, the degree of non-specific bindingexhibited by the disclosed low-binding surfaces may be assessed using astandardized protocol for contacting the surface with a labeled protein(e.g., bovine serum albumin (BSA), streptavidin, a DNA polymerase, areverse transcriptase, a helicase, a single-stranded binding protein(SSB), etc., or any combination thereof), a labeled nucleotide, alabeled oligonucleotide, etc., under a standardized set of incubationand rinse conditions, followed by detection of the amount of labelremaining on the surface and comparison of the signal resultingtherefrom to an appropriate calibration standard. In some instances, thelabel may comprise a fluorescent label. In some instances, the label maycomprise a radioisotope. In some instances, the label may comprise anyother detectable label. In some instances, the degree of non-specificbinding exhibited by a given surface formulation may thus be assessed interms of the number of non-specifically bound protein molecules (orother molecules) per unit area. In some instances, the low-bindingsurfaces of the present disclosure may exhibit non-specific proteinbinding (or non-specific binding of other specified molecules, e.g., Cy3dye) of less than or equal to about 0.001 molecule per μm², less than orequal to about 0.01 molecule per μm², less than or equal to about 0.1molecule per μm², less than or equal to about 0.25 molecule per μm²,less than or equal to about 0.5 molecule per μm², less than or equal toabout 1 molecule per μm², less than or equal to about 10 molecules perμm², less than or equal to about 100 molecules per μm², or less than orequal to about 1,000 molecules per μm². A given surface of the presentdisclosure may exhibit non-specific binding falling anywhere within thisrange, for example, of less than or equal to about 86 molecules per μm².For example, some modified surfaces disclosed herein exhibitnon-specific protein binding of less than or equal to about 0.5molecule/μm² following contact with a 1 μM solution of bovine serumalbumin (BSA) in phosphate buffered saline (PBS) buffer for 30 minutes,followed by a 10 minute PBS rinse. In another example, some modifiedsurfaces disclosed herein exhibit non-specific protein binding of lessthan or equal to about 0.5 molecule/μm² following contact with a 1 μMsolution of Cyanine 3 dye-labeled streptavidin (GE Amersham) inphosphate buffered saline (PBS) buffer for 15 minutes, followed by 3rinses with deionized water. Some modified surfaces disclosed hereinexhibit non-specific binding of Cy3 dye molecules of less than or equalto about 0.25 molecules per μm².

The low-background surfaces consistent with the disclosure herein mayexhibit specific dye attachment (e.g., Cy3 attachment) to non-specificdye adsorption (e.g., Cy3 dye adsorption) ratios of at least 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50specific dye molecules attached per molecule non-specifically adsorbed.Similarly, when subjected to an excitation energy, low-backgroundsurfaces consistent with the disclosure herein to which fluorophores,e.g., Cy3, have been attached may exhibit ratios of specificfluorescence signal (e.g., arising from Cy3-labeled oligonucleotidesattached to the surface) to non-specific adsorbed dye fluorescencesignals of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1,30:1, 40:1, 50:1, or more than 50:1.

In some instances, the degree of hydrophilicity (or “wettability” withaqueous solutions) of the disclosed surfaces may be assessed, forexample, through the measurement of water contact angles in which asmall droplet of water is placed on the surface and its angle of contactwith the surface is measured using, e.g., an optical tensiometer. Insome instances, a static contact angle may be determined. In someinstances, an advancing or receding contact angle may be determined. Insome instances, the water contact angle for the hydrophilic, low-bindingsurfaces disclosed herein may range from about 0 degrees to about 30degrees. In some instances, the water contact angle for the hydrophilic,low-binding surfaced disclosed herein may be no more than 50 degrees, 40degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2degrees, or 1 degree. In many cases the contact angle is no more than 40degrees. A given hydrophilic, low-binding surface of the presentdisclosure may exhibit a water contact angle having a value of anywherewithin this range.

In some instances, the low-binding surfaces of the present disclosuremay exhibit significant improvement in stability or durability toprolonged exposure to solvents and elevated temperatures, or to repeatedcycles of solvent exposure or changes in temperature. For example, insome instances, the stability of the disclosed surfaces may be tested byfluorescently labeling a functional group on the surface, or a tetheredbiomolecule (e.g., an oligonucleotide primer) on the surface, andmonitoring fluorescence signal before, during, and after prolongedexposure to solvents and elevated temperatures, or to repeated cycles ofsolvent exposure or changes in temperature. In some instances, thedegree of change in the fluorescence used to assess the quality of thesurface may be less than or equal to about 1%, 2%, 3%, 4%, 5%, 10%, 15%,20%, or 25% over a time period of 1 minute, 2 minutes, 3 minutes, 4minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30hours, 35 hours, 40 hours, 45 hours, 50 hours, or 100 hours of exposureto solvents, or, elevated temperatures, or a combination thereof (or anycombination of these percentages as measured over these time periods).In some instances, the degree of change in the fluorescence used toassess the quality of the surface may be less than or equal to about 1%,2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% over 5 cycles, 10 cycles, 20cycles, 30 cycles, 40 cycles, 50 cycles, 60 cycles, 70 cycles, 80cycles, 90 cycles, 100 cycles, 200 cycles, 300 cycles, 400 cycles, 500cycles, 600 cycles, 700 cycles, 800 cycles, 900 cycles, or 1,000 cyclesof repeated exposure to solvent changes, or, changes in temperature, ora combination thereof (or any combination of these percentages asmeasured over this range of cycles).

In some instances, the surfaces disclosed herein may exhibit a highratio of specific signal to non-specific signal or other background. Forexample, when used for nucleic acid amplification, some surfaces mayexhibit an amplification signal that is at least 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 75, 100, or greater than 100 fold greater than asignal of an adjacent unpopulated region of the surface. Similarly, somesurfaces exhibit an amplification signal that is at least 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 40, 50, 75, 100, or greater than 100 fold greaterthan a signal of an adjacent amplified nucleic acid population region ofthe surface.

Accordingly, low background surfaces as disclosed herein exhibit lowbackground fluorescence signals or high contrast to noise (CNR) ratios.

Flow Cell Devices: The low non-specific binding surfaces describedherein, in some examples, are surfaces of a flow device describedherein. Flow devices described herein can include a first reservoirhousing a first solution and having an inlet end and an outlet end,wherein the first agent flows from the inlet end to the outlet end inthe first reservoir; a second reservoir housing a second solution andhaving an inlet end and an outlet end, wherein the second agent flowsfrom the inlet end to the outlet end in the second reservoir; a centralregion having an inlet end fluidically coupled to the outlet end of thefirst reservoir and the outlet end of the second reservoir through atleast one valve. In the flow cell device, the volume of the firstsolution flowing from the outlet of the first reservoir to the inlet ofthe central region is less than the volume of the second solutionflowing from the outlet of the second reservoir to the inlet of thecentral region.

The reservoirs described in the device can be used to house differentreagents. In some examples, the first solution housed in the firstreservoir is different from the second solution that is housed in thesecond reservoir. The second solution comprises at least one reagentcommon to a plurality of reactions occurring in the central region. Insome examples, the second solution comprises at least one reagentselected from the list consisting of a solvent, a polymerase, and adNTP. In some examples, the second solution comprise low cost reagents.In some examples, the first reservoir is fluidically coupled to thecentral region through a first valve and the second reservoir isfluidically coupled to the central region through a second valve. Thevalve can be a diaphragm valve or other suitable valves.

The central region can include a capillary tube or microfluidic chiphaving one or more microfluidic channels. In some examples, thecapillary tube is an off-shelf product. The capillary tube or themicrofluidic chip can also be removable from the device. In someexamples, the capillary tube or microfluidic channel comprises anoligonucleotide population directed to sequence a eukaryotic genome. Insome examples, the capillary tube or microfluidic channel in the centralregion is removable.

Disclosed herein are single capillary flow cell devices that comprise asingle capillary and one or two fluidic adapters affixed to one or bothends of the capillary, where the capillary provides a fluid flow channelof specified cross-sectional area and length, and where the fluidicadapters are configured to mate with standard tubing to provide forconvenient, interchangeable fluid connections with an external fluidflow control system. In general, the capillary used in the disclosedflow cell devices (and flow cell cartridges to be described below) willhave at least one internal, axially-aligned fluid flow channel (or“lumen”) that runs the full length of the capillary. In some examples,the capillary may have two, three, four, five, or more than fiveinternal, axially-aligned fluid flow channels (or “lumens”).

A number specified cross-sectional geometries for a single capillary (ora lumen thereof) are consistent with the disclosure herein, including,but not limited to, circular, elliptical, square, rectangular,triangular, rounded square, rounded rectangular, or rounded triangularcross-sectional geometries. In some examples, the single capillary (orlumen thereof) may have any specified cross-sectional dimension or setof dimensions. For example, in some examples, the largestcross-sectional dimension of the capillary lumen (e.g., the diameter, ifthe lumen is circular in shape, or the diagonal, if the lumen is squareor rectangular in shape) may range from about 10 μm to about 10 mm. Thelength of the one or more capillaries used to fabricate the disclosedsingle capillary flow cell devices or flow cell cartridges may rangefrom about 5 mm to about 5 cm or greater. Capillaries, in some examples,have a gap height of about or exactly 50, 75, 100, 125, 150, 175, 200,225, 250, 275, 300, 350, 400, or 500 um, or any value falling within therange defined thereby.

Disclosed herein also are flow cell devices that comprise one or moremicrofluidic chips and one or two fluidic adapters affixed to one orboth ends of the microfluidic chips, where the microfluidic chipprovides one or more fluid flow channels of specified cross-sectionalarea and length, and where the fluidic adapters are configured to matewith the microfluidic chip to provide for convenient, interchangeablefluid connections with an external fluid flow control system.

The microfluidic chip described herein includes one or more microfluidicchannels etched on the surface of the chip. The microfluidic channelsare defined as fluid conduits with at least one minimum dimension from<1 nm to 1000 μm. The microfluidic channel system, fabricated on eithera glass or silicon substrate, has channel heights and widths on theorder of <1 nm to 1000 μm. The channel length can be in the micrometerrange.

The capillaries or microfluidic chip used for constructing the disclosedflow cell devices may be fabricated from any of a variety of materialsknown to those of skill in the art including, but not limited to, glass(e.g., borosilicate glass, soda lime glass, etc.), fused silica(quartz), polymer (e.g., polystyrene (PS), macroporous polystyrene(MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene(PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefinpolymers (COP), cyclic olefin copolymers (COC), polyethyleneterephthalate (PET), polydimethylsiloxane (PDMS), etc.), polyetherimide(PEI) and perfluoroelastomer (FFKM) as more chemically inert examples.PEI is somewhere between polycarbonate and PEEK in terms of both costand compatibility. FFKM is also known as Kalrez.

In some examples, a flow cell device described herein (e.g., amicrofluidic chip or capillary flow cell) is operatively coupled to animaging system described herein to capture or detect signals of DNAbases for applications such as nucleic acid sequencing, analyte captureand detection, and the like.

Oligonucleotide primers and adapter sequences: In general, at least onelayer of the one or more layers of low non-specific binding material maycomprise functional groups for covalently or non-covalently attachingoligonucleotide adapter or primer sequences, or the at least one layermay already comprise covalently or non-covalently attachedoligonucleotide adapter or primer sequences at the time that it isdeposited on the support surface. In some instances, theoligonucleotides tethered to the polymer molecules of at least one thirdlayer may be distributed at a plurality of depths throughout the layer.

One or more types of oligonucleotide primer may be attached or tetheredto the support surface. In some instances, the one or more types ofoligonucleotide adapters or primers may comprise spacer sequences,adapter sequences for hybridization to adapter-ligated template librarynucleic acid sequences, forward amplification primers, reverseamplification primers, sequencing primers, or, molecular barcodingsequences, or any combination thereof. In some instances, 1 primer oradapter sequence may be tethered to at least one layer of the surface.In some instances, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10different primer or adapter sequences may be tethered to at least onelayer of the surface.

In some instances, the tethered oligonucleotide adapter, or, primersequences, or a combination thereof may range in length from about 10nucleotides to about 100 nucleotides. In some instances, the tetheredoligonucleotide adapter, or, primer sequences, or a combination thereofmay be at least 10, at least 20, at least 30, at least 40, at least 50,at least 60, at least 70, at least 80, at least 90, or at least 100nucleotides in length. In some instances, the tethered oligonucleotideadapter, or, primer sequences, or a combination thereof may be at most100, at most 90, at most 80, at most 70, at most 60, at most 50, at most40, at most 30, at most 20, or at most 10 nucleotides in length. Any ofthe lower and upper values described in this paragraph may be combinedto form a range included within the present disclosure, for example, insome instances the length of the tethered oligonucleotide adapter, or,primer sequences, or combination thereof may range from about 20nucleotides to about 80 nucleotides. The length of the tetheredoligonucleotide adapter, or, primer sequences, or combination thereofmay have any value within this range, e.g., about 24 nucleotides.

In some instances, the tethered primer sequences may comprisemodifications designed to facilitate the specificity and efficiency ofnucleic acid amplification as performed on the low-binding supports. Forexample, in some instances the primer may comprise polymerase stoppoints such that the stretch of primer sequence between the surfaceconjugation point and the modification site is always in single-strandedform and functions as a loading site for 5′ to 3′ helicases in somehelicase-dependent isothermal amplification methods. Other examples ofprimer modifications that may be used to create polymerase stop pointsinclude, but are not limited to, an insertion of a PEG chain into thebackbone of the primer between two nucleotides towards the 5′ end,insertion of an abasic nucleotide (e.g., a nucleotide that has neither apurine nor a pyrimidine base), or a lesion site which can be bypassed bythe helicase.

As will be discussed further in the examples below, the surface densityof tethered primers on the support surface, or, the spacing of thetethered primers away from the support surface (e.g., by varying thelength of a linker molecule used to tether the primers to the surface),or a combination thereof, may be varied in order to “tune” the supportfor optimal performance when using a given amplification method. Asnoted below, adjusting the surface density of tethered primers mayimpact the level of specific, or, non-specific amplification, or acombination thereof, observed on the support in a manner that variesaccording to the amplification method selected. In some instances, thesurface density of tethered oligonucleotide primers may be varied byadjusting the ratio of molecular components used to create the supportsurface. For example, in an example where an oligonucleotide primer—PEGconjugate is used to create the final layer of a low-binding support,the ratio of the oligonucleotide primer—PEG conjugate to anon-conjugated PEG molecule may be varied. The resulting surface densityof tethered primer molecules may then be estimated or measured using anyof a variety of techniques. Examples include, but are not limited to,the use of radioisotope labeling and counting methods, covalent couplingof a cleavable molecule that comprises an optically-detectable tag(e.g., a fluorescent tag) that may be cleaved from a support surface ofdefined area, collected in a fixed volume of an appropriate solvent, andthen quantified by comparison of fluorescence signals to that for acalibration solution of known optical tag concentration, or usingfluorescence imaging techniques provided that care has been taken withthe labeling reaction conditions and image acquisition settings toensure that the fluorescence signals are linearly related to the numberof fluorophores on the surface (e.g., that there is no significantself-quenching of the fluorophores on the surface).

In some instances, the resultant surface density of oligonucleotideprimers on the low binding support surfaces of the present disclosuremay range from about 1,000 primer molecules per μm² to about 1,000,000primer molecules per μm². In some instances, the surface density ofoligonucleotide primers may be at least 1,000, at least 10,000, at least100,000, or at least 1,000,000 molecules per μm². In some instances, thesurface density of oligonucleotide primers may be at most 1,000,000, atmost 100,000, at most 10,000, or at most 1,000 molecules per μm². Any ofthe lower and upper values described in this paragraph may be combinedto form a range included within the present disclosure, for example, insome instances the surface density of primers may range from about10,000 molecules per μm² to about 100,000 molecules per μm². The surfacedensity of primer molecules may have any value within this range, e.g.,about 455,000 molecules per μm². In some instances, the surface densityof template library nucleic acid sequences initially hybridized toadapter or primer sequences on the support surface may be less than orequal to that indicated for the surface density of tetheredoligonucleotide primers. In some instances, the surface density ofclonally-amplified template library nucleic acid sequences hybridized toadapter or primer sequences on the support surface may span the samerange as that indicated for the surface density of tetheredoligonucleotide primers.

Local densities as listed above do not preclude variation in densityacross a surface, such that a surface may comprise a region having anoligo density of, for example, 500,000 per μm², while also comprising atleast a second region having a substantially different local density.

Imaging Systems. Imaging systems described herein are utilized to detecthybridization between one or more sample nucleic acid molecules andcapture nucleic acid molecules coupled to a low non-specific bindingsurface. In some examples, the imaging systems comprise a camera. Insome examples, the imaging systems comprise a microscope, such as afluorescence microscope. An inverted fluorescence microscope incombination with a camera may be used to capture an image of the lownon-specific binding surface and visualize hybridization between one ormore sample nucleic acid molecules and capture nucleic acid molecules. Anon-limiting example of an imaging system described herein is an OlympusIX83 microscope (Olympus Corp., Center Valley, Pa.) with a totalinternal reflectance fluorescence (TIRF) objective (100×, 1.5 NA,Olympus), a CCD camera (e.g., an Olympus EM-CCD monochrome camera,Olympus XM-10 monochrome camera, or an Olympus DP80 color and monochromecamera), an illumination source (e.g., an Olympus 100W Hg lamp, anOlympus 75W Xe lamp, or an Olympus U-HGLGPS fluorescence light source),and excitation wavelengths of 532 nm or 635 nm. Dichroic mirrors werepurchased from Semrock (IDEX Health & Science, LLC, Rochester, N.Y.),e.g., 405, 488, 532, or 633 nm dichroic reflectors/beamsplitters, andband pass filters were chosen as 532 LP or 645 LP concordant with theappropriate excitation wavelength.

Computer Control Systems. The present disclosure provides computersystems that are programmed or otherwise configured to implement methodsprovided herein, such as, for example, methods for nucleic sequencing,storing reference nucleic acid sequences, conducting sequence analysisand/or comparing sample and reference nucleic acid sequences asdescribed herein. An example of such a computer system is shown in FIG.10. As shown in FIG. 10, the computer system 1001 includes a centralprocessing unit (CPU, also “processor” and “computer processor” herein)1005, which can be a single core or multi core processor, or a pluralityof processors for parallel processing. The computer system 1001 alsoincludes memory or memory location 1010 (e.g., random-access memory,read-only memory, flash memory), an electronic storage unit 1015 (e.g.,hard disk), a communication interface 1020 (e.g., network adapter) forcommunicating with one or more other systems, and peripheral devices1025, such as cache, other memory, data storage and/or electronicdisplay adapters. The memory 1010, storage unit 1015, interface 1020 andperipheral devices 1025 are in communication with the CPU 1005 through acommunication bus (solid lines), such as a motherboard. The storage unit1015 can be a data storage unit (or data repository) for storing data.The computer system 1001 can be operatively coupled to a computernetwork (“network”) 1030 with the aid of the communication interface1020. The network 1030 can be the Internet, an internet and/or extranet,or an intranet and/or extranet that is in communication with theInternet. The network 1030 in some cases is a telecommunication and/ordata network. The network 1030 can include one or more computer servers,which can enable distributed computing, such as cloud computing. Thenetwork 1030, in some instances, with the aid of the computer system1001, can implement a peer-to-peer network, which may enable devicescoupled to the computer system 1001 to behave as a client or a server.

The CPU 1005 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1010. Examples ofoperations performed by the CPU 1005 can include fetch, decode, execute,and writeback.

The storage unit 1015 can store files, such as drivers, libraries andsaved programs. The storage unit 1015 can store user data, e.g., userpreferences and user programs. The computer system 1001 in someinstances can include one or more additional data storage units that areexternal to the computer system 1001, such as located on a remote serverthat is in communication with the computer system 1001 through anintranet or the Internet.

The computer system 1001 can communicate with one or more remotecomputer systems through the network 1030. For instance, the computersystem 1001 can communicate with a remote computer system of a user(e.g., operator). Examples of remote computer systems include personalcomputers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad,Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone,Android-enabled device, Blackberry®), or personal digital assistants.The user can access the computer system 1001 via the network 1030.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1001, such as, for example, on thememory 1010 or electronic storage unit 1015. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1005. In some cases, thecode can be retrieved from the storage unit 1015 and stored on thememory 1010 for ready access by the processor 1005. In some situations,the electronic storage unit 1015 can be precluded, andmachine-executable instructions are stored on memory 1010.

The code can be pre-compiled and configured for use with a machinecomprising a processer adapted to execute the code or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 1001, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such memory (e.g., read-only memory, random-access memory,flash memory) or a hard disk. “Storage” type media can include any orall of the tangible memory of the computers, processors or the like, orassociated modules thereof, such as various semiconductor memories, tapedrives, disk drives and the like, which may provide non-transitorystorage at any time for the software programming. All, or portions, ofthe software may at times be communicated through the Internet orvarious other telecommunication networks. Such communications, forexample, may enable loading of the software from one computer orprocessor into another, for example, from a management server or hostcomputer into the computer platform of an application server. Thus,another type of media that may bear the software elements includesoptical, electrical and electromagnetic waves, such as those used acrossphysical interfaces between local devices, through wired and opticallandline networks and over various air-links. The physical elements thatcarry such waves, such as wired or wireless links, optical links or thelike, also may be considered as media bearing the software. As usedherein, unless restricted to non-transitory, tangible “storage” media,terms such as computer or machine “readable medium” refer to any mediumthat participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include, for example, dynamic memory,such as main memory of such a computer platform. Tangible transmissionmedia include, for example, coaxial cables; copper wire and fiberoptics, including the wires that comprise a bus within a computersystem. Carrier-wave transmission media may take the form of electric orelectromagnetic signals, or acoustic or light waves such as thosegenerated during radio frequency (RF) and infrared (IR) datacommunications. Common forms of computer-readable media thereforeinclude, for example: a floppy disk, a flexible disk, a hard disk,magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, anyother optical medium, punch cards paper tape, any other physical storagemedium with patterns of holes, a RAM, a ROM, a PROM and EPROM, aFLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1001 can include, or be in communication with, anelectronic display 1035 that comprises a user interface (UI) forproviding, for example, an output or readout of a nucleic acidsequencing instrument coupled to the computer system 1001. Such readoutcan include a nucleic acid sequencing readout, such as a sequence ofnucleic acid bases that comprise a given nucleic acid sample. The UI mayalso be used to display the results of an analysis making use of suchreadout. Examples of UI's include, without limitation, a graphical userinterface (GUI) and web-based user interface. The electronic display1035 can be a computer monitor, or a capacitive or resistivetouchscreen.

Performance of Compositions and Systems

Improvements in hybridization rate: In some instances, the use of thebuffer formulations disclosed herein (optionally, used in combinationwith a low non-specific binding surface) yield relative hybridizationrates that range from about 2× to about 20× faster than that for astandard hybridization protocol. In some instances, the relativehybridization rate may be at least 2×, at least 3×, at least 4×, atleast 5×, at least 6×, at least 7×, at least 8×, at least 9×, at least10×, at least 12×, at least 14×, at least 16×, at least 18×, or at least20× that for a standard hybridization protocol.

The method and compositions described herein can help shorten the timerequired for completing hybridization. In some embodiments, thehybridization time can be in the range of about 1 seconds (s) to 2 hours(h), about 5s to 1.5h, about 15s to 1h, or about 15s to 0.5h. In someembodiments, the hybridization time can be in the range of about 15s to1h. In some embodiments, the hybridization time can be shorter than 15s,30s, 1 minutes (min), 1.5 min, 2 min, 2.5 min, 3 min, 4 min, 5 min, 6min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, or 120min. In some embodiments, the hybridization time can be longer than 1s,5s, 10s, 15s, 30s, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 4 min, or 5min.

The annealing methods described herein can significantly shorten theannealing time. In some instances, at least 90% of the target nucleicacid anneals to the surface bound nucleic acid in less than or equal toabout 15s, 30s, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 4 min, 5 min, 6min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, or 120min. In some instances, at least 80% of the target nucleic acid annealsto the surface bound nucleic acid in less than or equal to about 15s,30s, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 4 min, 5 min, 6 min, 7 min,8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, 50 min, 60min, 70 min, 80 min, 90 min, 100 min, 110 min, or 120 min. In someinstances, at least 90% of the target nucleic acid anneals to thesurface bound nucleic acid in greater than or equal to about 1s, 5s,10s, 15s, 30s, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 4 min, or 5 min.In some instances, at least 90% of the target nucleic acid anneals tothe surface bound nucleic acid in the range of about 10s to about 1hour, about 30s to about 50 min, about 1 min to about 50 min, or about 1min to about 30 min. In some instances, at least 90% of the targetnucleic acid anneals to the surface bound nucleic acid in between 2-25,3-24, 4-23, 5-23, 6-22, 7-21, 8-20, 9-19, 10-18, 11-17, 12-16, or 13-15min.

Improvements in hybridization efficiency: As used herein, hybridizationefficiency (or yield) is a measure of the percentage of total availabletethered adapter sequences on a solid surface, primer sequences, oroligonucleotide sequences in general that are hybridized tocomplementary sequences. In some instances, the use of optimized bufferformulations disclosed herein (optionally, used in combination with alow non-specific binding surface) yield improved hybridizationefficiency compared to that for a standard hybridization protocol. Insome instances, the hybridization efficiency that may be achieved isbetter than 80%, 85%, 90%, 95%, 98%, or 99% in any of the hybridizationreaction times specified above.

The methods and compositions described herein can be used in anisothermal annealing conditions. In some embodiments, the methodsdescribed herein can eliminate the cooling required for mosthybridizations. In some embodiments, the annealing methods describedherein can be performed at a temperature in the range of about 10° C. to95° C., about 20° C. to 80° C., or about 30° C. to 70° C. In someembodiments, the temperature can be lower than about 40° C., 50° C., 60°C., 70° C., 80° C., or 90° C.

Improvements in hybridization specificity: Methods, systems,compositions, and kits described herein provide for improvedhybridization specificity, as compared to, for example, a hybridizationreaction performed on a low-non-specific binding surface describedherein at 90 degrees Celsius for 5 minutes followed by cooling for 120minutes to reach a final temperature of 37 degrees Celsius in a buffercomprising saline-sodium citrate. In some instances, the hybridizationspecificity that may be achieved is better than 1 base mismatch in 10hybridization events, 1 base mismatch in 100 hybridization events, 1base mismatch in 1,000 hybridization events, or 1 base mismatch in10,000 hybridization events. Hybridization specificity may be measuredusing techniques described herein

In some examples, at least or about 70%, 80%, or 90% of the samplenucleic acid molecules correctly hybridize to the capture nucleic acidmolecules (e.g., adapter sequences, primer sequences, or oligonucleotidesequences) with a complementary sequence. In some examples, more than90% of the sample nucleic acid molecules correctly hybridize to thecapture nucleic acid molecules. In some examples, between 90%-99% of thesample nucleic acid molecules correctly hybridize to the capture nucleicacid molecules. In some examples, 100% of the sample nucleic acidmolecules correctly hybridize to the capture nucleic acid molecules.

Hybridization specificity can be measured, by hybridizing labeled (e.g.,Cy3) complementary oligos to surface bound nucleic acid moleculesimmobilized to the surface, dehybridizing and collecting the hybridizedoligos, measuring a fluorescent signal from the collected oligos using afluorescence plate reader at the appropriate excitation and emissionwavelengths (e.g., 532, peak 570/30). The results can be used to developstandard curves for accurately measuring concentration. This assay canbe repeated with oligos that show varying degrees of complementarity andthe respective specificities.

Hybridization specificity as measured on the surface, may be measured bydividing the nonspecific background counts (e.g., calculated usingmethods provided in example 3) by the nonspecific probehybridization-nonspecific background counts (also may be calculatedusing methods in example 3). Calibration curves can be built using thehybridization specificity measurements. Experiments with oligos havingvarying degrees of complementarity can be used to calculate respectivespecificities more accurately.

The specificity of a given nucleic acid probe, p, can be quantified bythe relative sensitivity when a p spot is exposed to a perfectly matchedtarget, t, or to a mismatch, m,

$\frac{{}_{}^{}{}_{}^{}}{{}_{}^{}{}_{}^{}} = {\frac{{}_{\;}^{}{}_{}^{}}{{}_{\;}^{}{}_{}^{}} = {\frac{K_{t}}{K_{m}}.}}$

The specificity of the assay can be quantified by considering thefraction of incorrectly hybridized probes, P_(m).

$P_{m} = {\frac{y}{x + y} = {\frac{c_{m}K_{m}}{{c_{m}K_{m}} + {c_{t}K_{t}}}.}}$

In this case, y=x(c_(m)/c_(t))(K_(m)/K_(t)).

Improvements in hybridization sensitivity. “Hybridization sensitivity”refers to a concentration range of sample (or target) nucleic moleculesin which hybridization occurs with a target hybridization specificity.In some instances, the target hybridization specificity is 90%, or more.In some instances, the methods, systems, compositions, and kitsdescribed herein utilize less than a 10 nanomolar concentration ofsample nucleic acid molecules to hybridize the sample nucleic acidmolecules to capture nucleic acid molecules with high specificity. Insome instances, between a 10 nanomolar and 50 picomolar concentration ofsample nucleic acid molecules is used. In some instances, between a 9nanomolar and 100 picomolar concentration of sample nucleic acidmolecules is used. In some instances, between a 9 nanomolar and 150picomolar concentration of sample nucleic acid molecules is used. Insome instances, between a 7 nanomolar and 200 picomolar concentration ofsample nucleic acid molecules is used. In some instances, between a 6nanomolar and 250 picomolar concentration of sample nucleic acidmolecules is used. In some instances, between a 5 nanomolar and 250picomolar concentration of sample nucleic acid molecules is used. Insome instances, between a 4 nanomolar and 300 picomolar concentration ofsample nucleic acid molecules is used. In some instances, between a 3nanomolar and 350 picomolar concentration of sample nucleic acidmolecules is used. In some instances, between a 2 nanomolar and 400picomolar concentration of sample nucleic acid molecules is used. Insome instances, between a 1 nanomolar and 500 picomolar concentration ofsample nucleic acid molecules is used. In some instances, less than orequal to about a 1 nanomolar concentration of sample nucleic acidmolecules is used. In some instances, less than or equal to about a 250picomolar concentration of sample nucleic acid molecules is used. Insome instances, less than or equal to about a 200 picomolarconcentration of a sample nucleic acid molecules is used. In someinstances, less than or equal to about a 150 picomolar concentration ofsample nucleic acid molecules is used. In some instances, less than orequal to about a 100 picomolar concentration of sample nucleic acidmolecules is used. In some instances, less than or equal to about a 50picomolar concentration of sample nucleic acid molecules is used.

In some instances, the hybridization sensitivity is calculated using theInternational Union of Pure and Applied Chemistry (IUPAC) analyticaltechniques, which identify the sensitivity, Se, with the slope of thecalibration curve. The calibration curve describes the measuredresponse, R, to a target concentration, c_(t), R(c_(t)), andS_(e)=dR/dc_(t).

The quantitative resolution of the assay, Δc_(t), is then specified byΔc_(t)=∈_(r)(c_(t))/S_(e)(c_(t)), where ∈_(r) is the measurement erroras given by its standard deviation. The detection limit, the lowestdetectable c_(t), is determined by Δc_(t)(c_(t)=0), because when theconcentration c_(t) is lower than Δc_(t)(c_(t)=0), the error is largerthan the signal; and assuming that R(ct) is proportional to theequilibrium hybridization fraction at the surface, x; i.e.,R(ct)=κx+const where κ is a constant. This assumption is justified whenthe following conditions are fulfilled: (1) nonspecific adsorption isnegligible and R is due only to hybridization at the surface; (2) theduration of the experiment is sufficiently long to allow thehybridization to reach equilibrium; and (3), the measured signal dependslinearly on the amount of oligonucleotides at the surface.

Nucleic Acid Sequencing Applications

Nucleic acid sequencing is among the many applications for which themethods, compositions, systems, and kits described herein may be useful.Referring to FIG. 2, the methods disclosed herein, in some embodiments,comprise preparing a library of sample nucleic acid molecules forsequencing, hybridizing the library of sample nucleic acids to nucleicacid molecules coupled to a low non-specific binding surface in thepresence of the hybridizing compositions described herein, amplifyingthe library of sample nucleic acids in situ, optionally linearizing theamplified sample nucleic acids in situ, de-hybridizing the linearizedand amplified sample nucleic acids from the nucleic acid moleculescoupled to the low non-specific binding surface, hybridizing a primersequence to the sample nucleic acids, and sequencing the sample nucleicacids.

FIG. 6 provides an example of a workflow of the methods describedherein, wherein a library of sample nucleic acid molecules is prepared601, for example by a split ligation protocol, the library of samplenucleic acid molecules is hybridized to nucleic acid molecules coupledto a low non-specific binding surface in the presence of a hybridizationcomposition described herein 602, hybridization of the sample nucleicacid molecules to the nucleic acid molecules coupled to the lownon-specific binding surface occurs 603, sequencing primers arehybridized to complementary primer binding sequences on sample nucleicacids 604, and sequencing of the sample nucleic acids is performed 605.

FIG. 7 provides an example sequencing workflow of the methods describedherein, wherein a labeled deoxyribonucleotide triphosphate (dNTP) bindsto the sample nucleic acid molecule to determine the identity of thecomplementary nucleotide in the nucleic acid sequence of the samplenucleic acid molecule 701. In some instances, the dNTP is labeled with afluorophore (e.g., Cy3), either directly or by interaction with alabeled detection reagent. The surface is optionally washed, to removethe unbound labeled dNTP. The surface is imaged to detect the presenceof the labeled dNTP 702. The labeled dNTP is unbound from the samplenucleic acid molecule, and a blocked unlabeled dNTP is incorporated intothe sample nucleic acid molecule 703. The blocked unlabeled nucleotideis cleaved 704. Steps 701-704 are repeated for the next nucleotide inthe sample nucleic acid molecule 705.

The methods, compositions, systems, and kits described herein provide atleast the following advantages, particularly in a nucleic acidsequencing process: (i) decreased fluidic wash times (due to reducednon-specific binding, and thus faster sequencing cycle times), (ii)decreased imaging times (and thus faster turnaround times for assayreadout and sequencing cycles), (iii) decreased overall work flow timerequirements (due to decreased cycle times), (iv) decreased detectioninstrumentation costs (due to the improvements in contrast-to-noiseratio), (v) improved readout (base-calling) accuracy (due toimprovements in contrast-to-noise ratio), (vi) improved reagentstability and decreased reagent usage requirements (and thus reducedreagents costs), and (vii) fewer run-time failures due to nucleic acidamplification failures.

Methods of Analyzing a Target Nucleic Acid Utilizing Multivalent Bindingor Incorporation Compositions. Disclosed herein are multivalent bindingor incorporation compositions and uses of said compositions in analyzingnucleic acid molecules, including in sequencing or other bioassayapplications. An increase in binding or incorporation of a nucleotide toan enzyme (e.g., polymerase) or an enzyme complex can be affected byincreasing the effective concentration of the nucleotide. The increasecan be achieved by increasing the concentration of the nucleotide infree solution, or by increasing the amount of the nucleotide inproximity to the relevant binding or incorporation site. The increasecan also be achieved by physically restricting a number of nucleotidesto a limited volume, thus resulting in a local increase inconcentration, and, resultingly, in the nucleotides binding orincorporating to a binding or incorporation site with a higher apparentavidity than would be observed with unconjugated, untethered, orotherwise unrestricted individual nucleotides. One non-limitingmechanism of effecting such a restriction is a multivalent binding orincorporation composition in which multiple nucleotides are bound to aparticle such as a polymer, a branched polymer, a dendrimer, a micelle,a liposome, a microparticle, a nanoparticle, a quantum dot, or othersuitable particle known in the art.

The multivalent binding or incorporation composition disclosed hereincan include at least one particle-nucleotide conjugate, wherein theparticle-nucleotide conjugate comprises a plurality of copies of thesame nucleotide attached to a particle. When the nucleotide iscomplementary to the target nucleic acid, the particle-nucleotideconjugate forms a binding or incorporation complex with the polymeraseand the target nucleic acid, and the binding or incorporation complexexhibits increased stability and longer persistence time than thebinding or incorporation complex formed using a single unconjugated oruntethered nucleotide. Each of the nucleotide moieties of themultivalent binding composition may bind to a complementary N+1nucleotide of a primed target nucleic acid molecule, thereby forming amultivalent binding complex comprising two or more target nucleic acidmolecules, two or more polymerase (or other enzyme) molecules, and themultivalent binding composition (e.g., the polymer-nucleotideconjugate). Each of the nucleotide moieties of the multivalent bindingcomposition may bind to a complementary N nucleotide of a primed targetnucleic acid molecule, thereby forming a multivalent binding complexcomprising two or more target nucleic acid molecules, two or morepolymerase (or other enzyme) molecules, and the multivalent bindingcomposition (e.g., the polymer-nucleotide conjugate). From this boundcomplex the nucleotide can interrogate the complementary base prior toincorporation of a modified reversibly blocked nucleotide that elongatesthe replicating strand by 1 base. In addition, it is possible to imagineinterrogation of the N nucleotide with a bound complex, stepping forwardwith a reversibly terminated nucleotide, and subsequently probing theN+1 base to pre- and post-deblocking. In this way you could performerror checking and improve the overall accuracy of base-calling byreading the interrogated base twice. The important discriminating factorfrom traditional methods is the binding is used to interrogate thematched base, while the stepping or incorporation step is used only tomove forward on the elongating strand.

The multivalent binding or incorporation composition described hereincan be used to localize detectable signals to active regions ofbiochemical interactions, such as sites of protein-nucleic acidinteractions, nucleic acid hybridization reactions, or enzymaticreactions, such as polymerase reactions. For instance, the multivalentbinding or incorporation composition described herein can be utilized toidentify sites of base incorporation in elongating nucleic acid chainsduring polymerase reactions and to provide base discrimination forsequencing and array-based applications. The increased binding orincorporation between the target nucleic acid and the nucleotide in themultivalent binding or incorporation composition, when the nucleotide iscomplementary to the target nucleic acid, provides enhanced signal thatgreatly improve base call accuracy and shortens imaging time.

In addition, the use of multivalent binding composition described hereinallows sequencing signals from a given sequence to originate withincluster regions containing multiple copies of the target sequence.Sequencing methods incorporating multiple copies of a target sequenceare advantageous in that signals can be amplified due to the presence ofmultiple simultaneous sequencing reactions within the defined region,each providing its own signal. The presence of multiple signals within adefined area also reduces the impact of any single skipped cycle, due tothe fact that the signal from a large number of correct base calls canoverwhelm the signal from a smaller number of skipped or incorrect basecalls, therefore providing methods for reducing phasing errors and/orimproving read length in sequencing reactions.

The multivalent binding compositions and their uses disclosed hereinlead to one or more of: (i) a stronger signal for better base-callingaccuracy compared to nucleic acid amplification and sequencingmethodologies using, for example, protic solvents; ii) greaterdiscrimination of sequence-specific signal from background signals;(iii) reduced requirements for the amount of starting materialnecessary, (iv) increased sequencing rate and shortened sequencing time;(v) reducing phasing errors, and (vi) improving read length insequencing reactions.

In some examples, the target nucleic acid refers to a target nucleicacid sample having one or more nucleic acid molecules. In some examples,the target nucleic acid includes a plurality of nucleic acid molecules.In some examples, the target nucleic acid includes two or more nucleicacid molecules. In some examples, the target nucleic acid includes twoor more nucleic acid molecules having the same sequences.

Sequencing Target Nucleic Acid

FIG. 12A-12H illustrate a non-limiting example of a method in which themultivalent binding composition is used for sequencing a target nucleiacid. As shown in FIG. 12A, the target nucleic acid 1202 can be tetheredto a solid support surface 1201. The target nucleic acid can be attachedto the surface either directly or indirectly. Although not shown in FIG.12A, the target nucleic acid 1202 can be hybridized to an adapter, whichis attached to the surface through a covalent or noncovalent bond. Whenone or more adapters are used to attach the target nucleic acid to thesurface, the target surface can comprise a fragment that iscomplementary to the adapter and thus hybridize to the adaptor. In someinstances, one adapter sequence may be tethered to the surface. In someinstances, a plurality of adapter sequences may be tethered to thesurface. In some instances, the target nucleic acid 1202 can also beattached directly to the solid-support surface without the use of anadapter. The solid support can be a low non-specific binding surface.

In FIG. 12B, after the initial step of attaching the target nucleic acidto the surface of a solid support surface (e.g., through hybridizationto adapters), the target nucleic acid is then clonally-amplified to formclusters of amplified nucleic acids. When the target nucleic acid isattached to the surface through an adapter, the surface density ofclonally-amplified nucleic acid sequences hybridized to adapter on thesupport surface may span the same range as the surface density oftethered adapters (or primers). The clonal amplification may beperformed using a polymerase chain reaction (PCR), multiple displacementamplification (MDA), transcription-mediated amplification (TMA), nucleicacid sequence-based amplification (NASBA), strand displacementamplification (SDA), real-time SDA, bridge amplification, isothermalbridge amplification, rolling circle amplification, circle-to-circleamplification, helicase-dependent amplification, recombinase-dependentamplification, single-stranded binding (SSB) protein-dependentamplification, or any combination thereof.

FIG. 12C illustrates a non-limiting step of annealing a primer 1203 tothe target nucleic acid 1202 to form a primed target nucleic acid 1204.FIG. 12B only shows one primer being used in the annealing step, butmore than one primer can be used depending on the types of targetnucleic acid. In some instances, the adapter that is used to attach thetarget nucleic acid to the surface has the same sequence as the primerused to prepare the primed target nucleic acid. The primer may compriseforward amplification primers, reverse amplification primers, sequencingprimers, and/or molecular barcoding sequences, or any combinationthereof. In some instances, one primer sequence may be used in thehybridization step. In some instances, a plurality of different primersequences may be used in the hybridization step.

As shown in FIG. 12D, the primed target nucleic acid 1204 is combinedwith a multivalent binding or incorporation composition and a polymerase1206 to form a binding or incorporation complex. The non-limitingexample of a multivalent binding or incorporation composition in FIG.12D comprises four particle-nucleotide conjugates 1205 a, 1205 b, 1205c, and 1205 d. Each particle-nucleotide conjugate has multiple copies ofa nucleotide attached to the particle, and the four particle-nucleotideconjugates cover four types of nucleotide respectively. Theparticle-nucleotide conjugate having a nucleotide that is complementaryto the next base on the primed target nucleic acid will form a bindingor incorporation complex with the polymerase and the target nucleicacid. In some instances, the multivalent binding or incorporationcomposition may include one, two or three particle-nucleotideconjugates. In some instances, each different type ofparticle-nucleotide conjugate can be labeled with a separate label. Insome instances, three of four types of nucleotide conjugates can belabeled, with a fourth either unlabeled or conjugated to an undetectablelabel. In some instances, 1, 2, 3, or 4 particle-nucleotide conjugatescan be labeled, either with the same label, or each with a labelcorresponding to the identity of its conjugated nucleotide, with,respectively, 3, 2, 1, or no particle-nucleotide conjugates that may beeither left unlabeled or conjugated to an undetectable label. In someexamples, detection of a polymerase complex incorporating aparticle-nucleotide conjugate may be carried out using four-colordetection, such that conjugates corresponding to all four nucleotidesare present in a sample, each conjugate having a separate labelcorresponding to the nucleotide conjugated thereto. In some examples,the four particle-nucleotide conjugates may be exposed to or contactedwith the target nucleic acid at the same time; in some other examples,the four particle-nucleotide conjugates may be exposed to or contactedwith the target nucleic acid sequentially, either individually, or ingroups of two or three. In some examples, detection of a polymerasecomplex incorporating a particle-nucleotide conjugate may be carried outusing three-color detection, such that conjugates corresponding to threeof the four nucleotides are present in a sample, with three conjugateshaving a separate label corresponding to the nucleotide conjugatedthereto and one conjugate having no label or being conjugated to anundetectable label. In some examples, only three types of conjugates areprovided, such that conjugates corresponding to three of the fournucleotides are present in a sample, with three conjugates having aseparate label corresponding to the nucleotide conjugated thereto andone conjugate being absent. In some examples, the identity ofnucleotides corresponding to an unlabeled or absent nucleotide conjugatecan be determined with respect to the location and/or identity of “dark”spots or locations of known target nucleic acids showing no fluorescencesignal. In some methods provided in the present disclosure, thedetection of the binding or incorporation complex is performed in theabsence of unbound or solution-borne polymer nucleotide conjugates.

In some examples, where three of the four particle-nucleotide conjugatesare labeled, or where only three of the four particle-nucleotideconjugates are present, the identity of the nucleotide corresponding tothe unlabeled or absent conjugate may be established by the absence of asignal or by monitoring of the presence of unlabeled complexes such asby the identification of “dark” spots or unlabeled regions in asequencing reaction. In some examples, detection of a polymerase complexincorporating a particle-nucleotide conjugate may be carried out usingtwo-color detection, such that conjugates corresponding to two of thefour nucleotides are present in a sample, with two conjugates having aseparate label corresponding to the nucleotide conjugated thereto andtwo conjugates having no label or being conjugated to an undetectablelabel. In some examples, only two of the four particle-nucleotideconjugates are labeled. In some examples, where two of the fourparticle-nucleotide conjugates are labeled, the identity of thenucleotide corresponding to the unlabeled conjugate or conjugates may beestablished by the absence of a signal or by monitoring of the presenceof unlabeled complexes such as by the identification of “dark” spots orunlabeled regions in a sequencing reaction. In some examples, where twoof the four particle-nucleotide conjugates are labeled, the fourparticle-nucleotide conjugates may be exposed to, or contacted with, thetarget nucleic acid sequentially, either individually, or in groups oftwo or three. In some examples, two of the four particle-nucleotideconjugates may share a common label, and the four particle-nucleotideconjugates may be exposed to or contacted with the target nucleic acidsequentially, either individually, or in groups of two or three, whereineach contacting step shows the distinction between two or more differentbases, such that after two, three, four, or more such contacting stepsthe identities of all unknown bases have been determined.

FIG. 12E illustrates the images captured on the surface after thebinding or incorporation complex is formed between the polymerase, thetarget nucleic acid, and the particle-nucleotide conjugate having anucleotide commentary to the next base of the primed target nucleicacid. The captured image includes four binding or incorporationcomplexes 1207 a, 1207 b, 1207 c, and 1207 d formed on the surface, andeach binding or incorporation complex has a different nucleotide whichcan be distinguished based on the label (e.g., fluorescence emissioncolor) on the particle-nucleotide conjugate. Because use of theparticle-nucleotide conjugate allows binding or incorporation signalsfrom a given sequence to originate within cluster regions containingmultiple copies of the target sequence, the sequencing signals aregreatly enhanced. Although FIG. 12E involves four particle-nucleotideconjugates, each having a different type of nucleotide, some methods canuse one, two, or three particle-nucleotide conjugates, each having adifferent type of nucleotide and label. In some examples, each differenttype of particle-nucleotide conjugate can be labeled either with thesame label, or each with a label corresponding to the identity of itsconjugated nucleotide. In some examples, three of four types ofnucleotide conjugates can be labeled, with a fourth either unlabeled orconjugated to an undetectable label. In some examples, 1, 2, 3, or 4particle-nucleotide conjugates can be labeled with a separate label,with, respectively, 3, 2, 1, or no particle-nucleotide conjugates eitherunlabeled or conjugated to an undetectable label. In some examples, adetection step can comprise simultaneous and/or serial excitation of upto 4 different excitation wavelengths, such as wherein the fluorescenceimaging is carried out by detecting single and/or multiple fluorescenceemission bands that uniquely classify each of the possible base pairings(A, G, C, or T). In some examples, four different nucleic acid bindingor incorporation compositions, each comprising a different nucleotide ornucleotide analog, may be used to determine the identity of the terminalnucleotide, wherein one of the four different nucleic acid binding orincorporation compositions is labeled with a first fluorophore, one islabeled with a second fluorophore, one is labeled with both the firstand second fluorophore, and one is not labeled, and wherein thedetecting step comprises simultaneous excitation at a first excitationwavelength and a second excitation wavelength and images are acquired ata first fluorescence emission wavelength and a second fluorescenceemission wavelength.

When the multivalent binding or incorporation composition is used inreplacement of single unconjugated or untethered nucleotides to form abinding or incorporation complex with the polymerase and the primedtarget nucleic acid, the local concentration of the nucleotide isincreased many-fold, which in turn enhances the signal intensity. Theformed binding or incorporation complex also has a longer persistencetime which in turn helps shorten the imaging step. The high signalintensity results from the high binding or incorporation avidity of thepolymer nucleotide conjugate (which may also comprise multiplefluorophores or other labels) which thus forms a complex which remainsstable for the entire binding or incorporation and imaging step. Thestrong binding or incorporation between the polymerase, the primedtarget strand, and the polymer-nucleotide or nucleotide analog conjugatealso means that the multivalent binding or incorporation complex thusformed will remain stable during washing steps, and the signal intensitywill remain high when other reaction mixture components and unmatchednucleotide analogs are washed away. After the imaging step, the bindingor incorporation complex can be destabilized (e.g., by changing thebuffer composition) and the primed target nucleic acid can then beextended for one base.

The sequencing method may further comprise incorporating the N+1 orterminal nucleotide into the primed strand as shown in FIG. 12F. In FIG.12F, the primer strand of the primed target nucleic acid 1208 can beextended for one base to form an extended nucleic acid 1209. Theextension step can occur after or concurrently with the destabilizationof the multivalent binding or incorporation complex. The primed targetnucleic acid 1208 can be extended using a complementary nucleotide thatis attached to the particle in the particle-nucleotide conjugate orusing an unconjugated or untethered free nucleotide that is providedafter the multivalent binding or incorporation composition has beenremoved.

After the extension step, the contacting step as shown in FIG. 12G canbe performed again to form binding or incorporation complexes andimitate the next sequencing cycle. The contacting, detecting, andextension steps can be repeated for one or more cycles, therebydetermining the sequence of the target nucleic acid molecule. Forexample, FIG. 12H illustrates the surface images obtained afterperforming multiple sequencing cycles, and the images can then beprocessed to determine the sequences of the target nucleic acidmolecules.

The extension of the primed target nucleic acid may be prevented orinhibited due to a blocked nucleotide on the strand or the use ofpolymerase that is catalytically inactive. When the nucleotide in thepolymer-nucleotide conjugate has a blocking group that prevents theextension of the nucleic acid, incorporation of a nucleotide may beachieved by the removal of a blocking group from said nucleotide (suchas by detachment of said nucleotide from its polymer, branched polymer,dendrimer, particle, or the like). When the extension of the primedtarget nucleic acid is inhibited due to the use of polymerase that iscatalytically inactive, incorporation of a nucleotide may be achieved bythe provision of a cofactor or activator such as a metal ion.

Also disclosed herein are systems configured for performing any of thedisclosed nucleic acid sequencing or nucleic acid analysis methods. Thesystem may comprise a fluid flow controller and/or fluid dispensingsystem configured to sequentially and iteratively contact the primedtarget nucleic acid molecules attached to a solid support with thedisclosed polymerase and multivalent binding or incorporationcompositions and/or reagents. The contacting may be performed within oneor more flow cells. In some instances, said flow cells may be fixedcomponents of the system. In some instances, said flow cells may beremovable and/or disposable components of the system.

The sequencing system may include an imaging module, i.e., one or morelight sources, one or more optical components, and one or more imagesensors for imaging and detection of binding or incorporation of thedisclosed nucleic acid binding or incorporation compositions to targetnucleic acid molecules tethered to a solid support or the interior of aflow cell. The disclosed compositions, reagents, and methods may be usedfor any of a variety of nucleic acid sequencing and analysisapplications. Examples include, but are not limited to, DNA sequencing,RNA sequencing, whole genome sequencing, targeted sequencing, exomesequencing, genotyping, and the like.

The sequencing system may also include computer control systems that areprogrammed to implement methods of the disclosure. The computer systemis programmed or otherwise configured to implement methods of thedisclosure including, for example, nucleic acid sequencing methods,interpretation of nucleic acid sequencing data and analysis of cellularnucleic acids, such as RNA (e.g., mRNA), or characterization of cellsfrom sequencing data. The computer system can be an electronic device ofa user or a computer system that is remotely located with respect to theelectronic device. The electronic device can be a mobile electronicdevice.

FIG. 13 is a flowchart outlining a non-limiting example of the steps insequencing a target nucleic acid. 1301 describes a step of attachingtarget library sequences to a solid support surface by hybridizing thetarget nucleic acid molecules to complementary adapters on a substratesurface. The target nucleic acid molecules can be single stranded orpartially double stranded. Prior to 1301, the nucleic acid molecules inthe target library may have been prepared to contain fragmentscomplementary to the adaptor sequences through ligation or othermethods. 1302 describes the step of clonal amplification to generateclusters of target nucleic acid molecules on the surface. 1303 describeshybridizing sequencing primers to complementary primer binding orincorporation sequences on the target nucleic acid to form the primedtarget nucleic acid. 1304 describes combining the polymerase, themultivalent binding or incorporation composition, which contains labeled(e.g., fluorescently-labeled) particle-nucleotide conjugates, and theprimed target nucleic acid. 1304 may also include a step of washing orremoving the unbound reagents including polymerase andparticle-nucleotide conjugate.

Again referring to FIG. 13, when the nucleotide on theparticle-nucleotide conjugate is complementary to the next base of theprimed target nucleic acid (1305), the particle-nucleotide conjugate,polymerase, and primed target nucleic acid form a ternary binding orincorporation complex, which can be detected by detection methods (e.g.,florescence imaging) compatible with the label on theparticle-nucleotide conjugate. 1305 can also include measuring thepersistence time of the ternary binding or incorporation complex. In1306, the binding or incorporation complex is destabilized to remove thebinding or incorporation of the particle-nucleotide conjugate andpolymerase. The dissociation can be achieved by placing the binding orincorporation complex in a condition (e.g., adding Strontium ions) thatwill change the conformation of the polymerase and destabilize thebinding or incorporation. 1306 may also include a step of washing orremoving the dissociated particle-nucleotide conjugate and/orpolymerase. 1307 describes the step of extending the primed strand ofthe primed target nucleic acid by a single base addition reaction. Afterthe single base extension, steps 1304, 1305, 1306, and 1307 can berepeated in multiple cycles to determine the sequences of the targetnucleic acid.

FIG. 14 is another flowchart outlining a non-limiting example of thesteps in sequencing a target nucleic acid, which includes cleaving anucleotide from the particle-nucleotide conjugate and incorporating thecleaved nucleotide. 1401 describes a step of attaching target librarysequences to a solid support surface by hybridizing the target nucleicacid molecules to complementary adapters on substrate surface. Thetarget nucleic acid molecules can be single stranded or partially doublestranded. Prior to 1401, the nucleic acid molecules in the targetlibrary may have been prepared to contain fragments complementary to theadaptor sequences through ligation or other methods. 1402 describes thestep of clonal amplification to generate clusters of target nucleic acidmolecules on the surface. 1403 describes hybridizing sequencing primersto complementary primer binding or incorporation sequences on the targetnucleic acid to form the primed target nucleic acid. 1404 describescombining the polymerase, the multivalent binding or incorporationcomposition, which contains labeled (e.g., fluorescently-labeled)particle-nucleotide conjugates, and the primed target nucleic acid. Inthe particle-nucleotide conjugates, the nucleotides are attached to theparticle through chemical bonds or interactions that can be latersevered. 1404 may also include a step of washing or removing the unboundreagents including polymerase and particle-nucleotide conjugate.

Again referring to FIG. 14, when the nucleotide on theparticle-nucleotide conjugate is complementary to the next base of theprimed target nucleic acid (1405), the particle-nucleotide conjugate,polymerase, and primed target nucleic acid form a ternary binding orincorporation complex, which can be detected by detection methods (e.g.,florescence imaging) compatible with the label on theparticle-nucleotide conjugate. 1405 can also include measuring thepersistence time of the ternary binding or incorporation complex. In1406, the polymerase is placed in a condition that would make itcatalytically active to incorporate a nucleotide. The condition caninclude exposing the polymerase to Mg or Mn ions in the reactionsolution. The nucleotide that is bound to the polymerase and the primedtarget nucleic acid is then cleaved from the particle and thenincorporated into the primed strand of the primed target nucleic acid.The binding or incorporation complex is destabilized. 1406 may alsoinclude a step of washing or removing the dissociatedparticle-nucleotide conjugate and/or polymerase. After the extension,steps 1404, 1405, and 1406 can be repeated in multiple cycles todetermine the sequences of the target nucleic acid.

Detecting Target Nucleic Acid Molecules. FIGS. 15A-15B illustrate oneexemplified method in which the multivalent binding or incorporationcomposition is used for detecting a target nucleic acid. As shown inFIG. 15A, the polymer-nucleotide conjugate 1501 is placed in contactwith polymerase 1506, a first nucleic acid molecule 1504, and a secondnucleic acid molecule 1505. The polymer-nucleotide conjugate 1501 hasmultiple polymer branches radiating from the core, and some branches areattached to a nucleotide or oligonucleotide 1502, and some branches areattached to a label 1503. When the nucleotide or oligonucleotide 1502 onthe polymer-nucleotide conjugate 1501 is complementary to at least afraction of the first nucleic acid 1504, a multivalent binding orincorporation complex is formed as shown in FIG. 15B, and the strongbinding or incorporation signal can help detect target nucleic acid withsequences complementary or partially complementary to the nucleotide oroligonucleotide on the polymer-nucleotide conjugate. In some instances,at least one of the polymerase, nucleic acid molecules, andpolymer-nucleotide conjugates is attached to a solid support.

The multivalent binding or incorporation composition described hereincan be used in a method of detecting a target nucleic acid in a sample.Also disclosed herein are systems configured for performing any of thedisclosed nucleic acid analysis methods. The systems may comprise afluid flow controller and/or fluid dispensing system configured tosequentially and iteratively contact the nucleic acid molecules with thedisclosed polymerase and multivalent binding or incorporationcompositions and/or reagents. The contacting may be performed within oneor more flow cells. In some instances, said flow cells may be fixedcomponents of the system. In some instances, said flow cells may beremovable and/or disposable components of the system. The system mayalso include a cartridge comprising a sample collection unit and anassay assembly, wherein the sample collection unit is configured tocollect a sample, and wherein the assay assembly comprises at least onereaction site containing a multivalent binding or incorporationcomposition adapted to interact with said analyte, allowing thepredetermined portion of sample to react with assay reagents containedwithin the assay assembly to yield a signal indicative of the presenceof the analyte in the sample, and detecting the signal generated fromthe analyte.

Multivalent Binding or incorporation Composition. The present disclosurerelates to multivalent binding or incorporation compositions having aplurality of nucleotides conjugated to a particle (e.g., a polymer,branched polymer, dendrimer, or equivalent structure). Contacting themultivalent binding or incorporation composition with a polymerase andmultiple copies of a primed target nucleic acid may result in theformation of a ternary complex which may be detected and in turn achievea more accurate determination of the bases of the target nucleic acid.

When the multivalent binding or incorporation composition is used inreplacement of a single unconjugated or untethered nucleotide to form acomplex with the polymerase and one or more copies of the target nucleicacid, the local concentration of the nucleotide as well as the bindingavidity of the complex (in the case that a complex comprising two ormore target nucleic acid molecules is formed) is increased many fold,which in turn enhances the signal intensity, particularly the correctsignal versus mismatch. The multivalent binding or incorporationcomposition described herein can include at least oneparticle-nucleotide conjugate (each particle-nucleotide conjugatecomprising multiple copies of a single nucleotide moiety) forinteracting with the target nucleic acid. The multivalent compositioncan also include two, three, or four different particle-nucleotideconjugates, each having a different nucleotide conjugated to theparticle.

The multivalent binding or incorporation composition can comprise 1, 2,3, 4, or more types of particle-nucleotide conjugates, wherein eachparticle-nucleotide conjugate comprises a different type of nucleotide.A first type of the particle-nucleotide conjugate can comprise anucleotide selected from the group consisting of ATP, ADP, AMP, dATP,dADP, and dAMP. A second type of the particle-nucleotide conjugate cancomprise a nucleotide selected from the group consisting of TTP, TDP,TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, and dUMP. A third typeof the particle-nucleotide conjugate can comprise a nucleotide selectedfrom the group consisting of CTP, CDP, CMP, dCTP, dCDP, and dCMP. Afourth type of the particle-nucleotide conjugate can comprise anucleotide selected from the group consisting of GTP, GDP, GMP, dGTP,dGDP, and dGMP. In some instances, each particle-nucleotide conjugatecomprises a single type of nucleotide respectively corresponding to oneor more nucleotides selected from the group consisting of ATP, ADP, AMP,dATP, dADP, dAMP TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP,dUDP, dUMP, CTP, CDP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, anddGMP. Each multivalent binding or incorporation composition may furthercomprise one or more labels corresponding to the particular nucleotideconjugated to each respective conjugate. Non-limiting examples of labelsinclude fluorescent labels, colorimetric labels, electrochemical labels(such as, for example, glucose or other reducing sugars, or thiols orother redox active moieties), luminescent labels, chemiluminescentlabels, spin labels, radioactive labels, steric labels, affinity tags,or the like.

Particle-Nucleotide Conjugate. In a particle-nucleotide conjugate,multiple copies of the same nucleotide may be covalently bound to ornoncovalently bound to the particle. Examples of the particle caninclude a branched polymer; a dendrimer; a cross linked polymer particlesuch as an agarose, polyacrylamide, acrylate, methacrylate,cyanoacrylate, methyl methacrylate particle; a glass particle; a ceramicparticle; a metal particle; a quantum dot; a liposome; an emulsionparticle, or any other particle (e.g, nanoparticles, microparticles, orthe like) known in the art. In one example, the particle is a branchedpolymer.

In some instances, the particle-nucleotide conjugate (e.g., apolymer-nucleotide conjugate) may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more than 10 copies of a nucleotide, nucleotide analog,nucleoside, or nucleoside analog tethered to the particle.

The nucleotide can be linked to the particle through a linker, and thenucleotide can be attached to one end or location of the polymer. Thenucleotide can be conjugated to the particle through the 5′ end of thenucleotide. In some particle-nucleotide conjugates, one nucleotide isattached to one end or location of a polymer. In someparticle-nucleotide conjugate, multiple nucleotides are attached to oneend or location of a polymer. The conjugated nucleotide is stericallyaccessible to one or more proteins, one or more enzymes, and nucleotidebinding or incorporation moieties. In some examples, a nucleotide may beprovided separately from a nucleotide binding or incorporation moietysuch as a polymerase. In some examples, the linker does not comprise aphoto emitting or photo absorbing group.

The particle can also have a binding or incorporation moiety. In someexamples, particles may self-associate without the use of a separateinteraction moiety. In some examples, particles may self-associate dueto buffer conditions or salt conditions, e.g., as in the case ofcalcium-mediated interactions of hydroxyapatite particles, lipid orpolymer mediated interactions of micelles or liposomes, or salt-mediatedaggregation of metallic (such as iron or gold) nanoparticles.

The particle-nucleotide conjugate can have one or more labels. Examplesof the labels include, but are not limited to, fluorophores, spinlabels, metals or metal ions, colorimetric labels, nanoparticles, PETlabels, radioactive labels, or other such labels as may render saidcomposition detectable by such methods as are known in the art of thedetection of macromolecules or molecular interactions. The label may beattached to the nucleotide (e.g., by attachment to the 5′ phosphatemoiety of a nucleotide), to the particle itself (e.g., to the PEGsubunits), to an end of the polymer, to a central moiety, or to anyother location within said polymer-nucleotide conjugate which would berecognized by one of skill in the art to be sufficient to render saidcomposition, such as a particle, detectable by such methods as are knownin the art or described elsewhere herein. In some examples, one or morelabels are provided so as to correspond to or differentiate a particularparticle-nucleotide conjugate.

In some examples, the label is a fluorophore. Non-limiting examples offluorescent moieties include, but are not limited to, fluorescein andfluorescein derivatives such as carboxyfluorescein,tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein,fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein,fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide,carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine andrhodamine derivatives such as TRITC, TMR, lissamine rhodamine, TexasRed, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine,TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissaminerhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Redhydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS,AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivativessuch as BODIPY FL C3-SE, BODIPY 530/550 C3, BODIPY 530/550 C3-SE, BODIPY530/550 C3 hydrazide, BODIPY 493/503 C3 hydrazide, BODIPY FL C3hydrazide, BODIPY FL IA, BODIPY 530/551 IA, Br-BODIPY 493/503, CascadeBlue and derivatives such as Cascade Blue acetyl azide, Cascade Bluecadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide,Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide,Lucifer Yellow CH, cyanine and derivatives such as indolium basedcyanine dyes, benzo-indolium based cyanine dyes, pyridium based cyaninedyes, thiozolium based cyanine dyes, quinolinium based cyanine dyes,imidazolium based cyanine dyes, Cy 3, Cy5, lanthanide chelates andderivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, Europium chelates,Terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCyclerRed dyes, CAL Flour dyes, JOE and derivatives thereof, Oregon Greendyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes,Malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes andothers known in the art such as those described in Haugland, MolecularProbes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles ofFluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), orHermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof,or any combination thereof. Cyanine dyes may exist in either sulfonatedor non-sulfonated forms, and consist of two indolenin, benzo-indolium,pyridium, thiozolium, and/or quinolinium groups separated by apolymethine bridge between two nitrogen atoms. Commercially availablecyanine fluorophores include, for example, Cy3, (which may comprise1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indoliumor1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate),Cy5 (which may comprise1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-iumor1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate),and Cy7 (which may comprise1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indoliumor1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate),where “Cy” stands for ‘cyanine’, and the first digit identifies thenumber of carbon atoms between two indolenine groups. Cy2, which is anoxazole derivative rather than indolenin, and the benzo-derivatizedCy3.5, Cy5.5 and Cy7.5 are exceptions to this rule.

In some embodiments, the detection label can be a FRET pair, such thatmultiple classifications can be performed under a single excitation andimaging step. As used herein, FRET may comprise excitation exchange(Forster) transfers, or electron-exchange (Dexter) transfers.

Polymer Nucleotide Conjugate. One example of the particle-nucleotideconjugate is a polymer-nucleotide conjugate. Some non-limiting examplesof the polymer-nucleotide conjugates are shown in FIGS. 16A-16C. Forexample, FIG. 16A shows polymer-nucleotide conjugates having variousconfigurations, e.g., a “starburst” configuration comprising afluorescently-labeled streptavidin core and four nucleotides bound tothe core via biotinylated, linear PEG linkers of molecular weightranging from 1K Dalton to 10K Daltons; FIG. 16B shows apolymer-nucleotide conjugate having a dendrimer core of, for example,12, 24, 48, or 96 arms, and linear PEG linkers of molecular weightranging from 1K Dalton to 10K Daltons radiating from the center; andFIG. 16C shows an example of polymer-nucleotide conjugates comprising anetwork of, e.g., streptavidin cores, linked together by branched PEGlinkers comprising a binding or incorporation moiety such as a biotin.

Examples of suitable linear or branched polymers include linear orbranched polyethylene glycol (PEG), linear or branched polypropyleneglycol, linear or branched polyvinyl alcohol, linear or branchedpolylactic acid, linear or branched polyglycolic acid, linear orbranched polyglycine, linear or branched polyvinyl acetate, a dextran,or other such polymers, or copolymers incorporating any two or more ofthe foregoing or incorporating other polymers as are known in the art.In one example, the polymer is a PEG. In another embodiment, the polymercan have PEG branches.

Suitable polymers may be characterized by a repeating unit incorporatinga functional group suitable for derivatization such as an amine, ahydroxyl, a carbonyl, or an allyl group. The polymer can also have oneor more pre-derivatized substituents such that one or more particularsubunits will incorporate a site of derivatization or a branch site,whether or not other subunits incorporate the same site, substituent, ormoiety. A pre-derivatized substituent may comprise or may furthercomprise, for example, a nucleotide, a nucleoside, a nucleotide analog,a label such as a fluorescent label, radioactive label, or spin label,an interaction moiety, an additional polymer moiety, or the like, or anycombination of the foregoing.

In the polymer-nucleotide conjugate, the polymer can have a plurality ofbranches. The branched polymer can have various configurations,including, but not limited to, stellate (“starburst”) forms, aggregatedstellate (“helter skelter”) forms, bottle brush, or dendrimer. Thebranched polymer can radiate from a central attachment point or centralmoiety, or may incorporate multiple branch points, such as, for example,2, 3, 4, 5, 6, 7, 8, 9, 10, or more branch points. In some instances,each subunit of a polymer may optionally constitute a separate branchpoint.

The length and size of the branch can differ based on the type ofpolymer. In some branched polymers, the branch may have a length ofbetween 1 and 1,000 nm, between 1 and 100 nm, between 1 and 200 nm,between 1 and 300 nm, between 1 and 400 nm, between 1 and 500 nm,between 1 and 600 nm, between 1 and 700 nm, between 1 and 800 nm, orbetween 1 and 900 nm, or more, or may have a length falling within orbetween any of the values disclosed herein.

In some polymer-nucleotide conjugates, the polymer core may have a sizecorresponding to an apparent molecular weight of 1K Da, 2K Da, 3K Da, 4KDa, 5K Da, 10K Da, 15K Da, 20K Da, 30K Da, 50K Da, 80K Da, 100K Da, orany value within a range defined by any two of the foregoing. Theapparent molecular weight of a polymer may be calculated from the knownmolecular weight of a representative number of subunits, as determinedby size exclusion chromatography, as determined by mass spectrometry, oras determined by any other method as is known in the art.

In some branched polymers, the branch may have a size corresponding toan apparent molecular weight of 1K Da, 2K Da, 3K Da, 4K Da, 5K Da, 10KDa, 15K Da, 20K Da, 30K Da, 50K Da, 80K Da, 100K Da, or any value withina range defined by any two of the foregoing. The apparent molecularweight of a polymer may be calculated from the known molecular weight ofa representative number of subunits, as determined by size exclusionchromatography, as determined by mass spectrometry, or as determined byany other method as is known in the art. The polymer can have multiplebranches. The number of branches in the polymer can be 2, 3, 4, 5, 6, 7,8, 12, 16, 24, 32, 64, 128 or more, or a number falling within a rangedefined by any two of these values.

For polymer-nucleotide conjugates comprising a branched polymer of, forexample, a branched PEG comprising 4, 8, 16, 32, or 64 branches, thepolymer nucleotide conjugate can have nucleotides attached to the endsof the PEG branches, such that each end has attached thereto 0, 1, 2, 3,4, 5, 6 or more nucleotides. In one non-limiting example, a branched PEGpolymer of between 3 and 128 PEG arms may have attached to the ends ofthe polymer branches one or more nucleotides, such that each end hasattached thereto 0, 1, 2, 3, 4, 5, 6 or more nucleotides or nucleotideanalogs. In some embodiments, a branched polymer or dendrimer has aneven number of arms. In some embodiments, a branched polymer ordendrimer has an odd number of arms.

In some instances, the length of the linker (e.g., a PEG linker) mayrange from about 1 nm to about 1,000 nm. In some instances, the lengthof the linker may be at least 1 nm, at least 10 nm, at least 25 nm, atleast 50 nm, at least 75 nm, at least 100 nm, at least 200 nm, at least300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700nm, at least 800 nm, at least 900 nm, or at least 1,000 nm. In someinstances, the length of the linker may range between any two of thevalues in this paragraph. For example, in some instances, the length ofthe linker may range from about 75 nm to about 400 nm. Those of skill inthe art will recognize that in some instances, the length of the linkermay have any value within the range of values in this paragraph, e.g.,834 nm.

In some instances, the length of the linker is different for differentnucleotides (including deoxyribonucleotides and ribonucleotides),nucleotide analogs (including deoxyribonucleotide analogs andribonucleotide analogs), nucleosides (including deoxyribonucleosides orribonucleosides), or nucleoside analogs (including deoxyribonucleosideanalogs or ribonucleoside analogs). In some instances, one of thenucleotides, nucleotide analogs, nucleosides, or nucleoside analogscomprises, for example, deoxyadenosine, and the length of the linker isbetween 1 nm and 1,000 nm. In some instances, one of the nucleotides,nucleotide analogs, nucleosides, or nucleoside analogs comprises, forexample, deoxyguanosine, and the length of the linker is between 1 nmand 1,000 nm. In some instances, one of the nucleotides, nucleotideanalogs, nucleosides, or nucleoside analogs comprises, for example,thymidine, and the length of the linker is between 1 nm and 1,000 nm. Insome instances, one of the nucleotides, nucleotide analogs, nucleosides,or nucleoside analogs comprises, for example, comprises deoxyuridine,and the length of the linker is between 1 nm and 1,000 nm. In someinstances, one of the nucleotides, nucleotide analogs, nucleosides, ornucleoside analogs comprises, for example, deoxycytidine, and the lengthof the linker is between 1 nm and 1,000 nm. In some instances, one ofthe nucleotides, nucleotide analogs, nucleosides, or nucleoside analogscomprises, for example, adenosine, and the length of the linker isbetween 1 nm and 1,000 nm. In some instances, one of the nucleotides,nucleotide analogs, nucleosides, or nucleoside analogs comprises, forexample, guanosine, and the length of the linker is between 1 and 1,000nm. In some instances, one of the nucleotides, nucleotide analogs,nucleosides, or nucleoside analogs comprises, for example,5-methyl-uridine, and the length of the linker is between 1 nm and 1,000nm. In some instances, one of the nucleotides, nucleotide analogs,nucleosides, or nucleoside analogs comprises, for example, uridine, andthe length of the linker is between 1 nm and 1,000 nm. In someinstances, one of the nucleotides, nucleotide analogs, nucleosides, ornucleoside analogs comprises, for example, cytidine, and the length ofthe linker is between 1 nm and 1,000 nm.

In the polymer-nucleotide conjugate, each branch or a subset of branchesof the polymer may have attached thereto a moiety comprising anucleotide (e.g., an adenine, a thymine, a uracil, a cytosine, or aguanine residue or a derivative or mimetic thereof), and the moiety iscapable of binding or incorporation to a polymerase, reversetranscriptase, or other nucleotide binding or incorporation domain.Optionally, the moiety may be capable of being incorporated into anelongating nucleic acid chain during a polymerase reaction. In someinstances, said moiety may be blocked such that it is not capable ofbeing incorporated into an elongating nucleic acid chain during apolymerase reaction. In some other instances, said moiety may bereversibly blocked such that it is not capable of being incorporatedinto an elongating nucleic acid chain during a polymerase reaction untilsuch block is removed, after which said moiety is then capable of beingincorporated into an elongating nucleic acid chain during a polymerasereaction.

The nucleotide can be conjugated to the polymer branch through the 5′end of the nucleotide. In some instances, the nucleotide may be modifiedso as to inhibit or prevent incorporation of the nucleotide into anelongating nucleic acid chain during a polymerase reaction. By way ofexample, the nucleotide may include a 3′ deoxyribonucleotide, a 3′azidonucleotide, a 3′-methyl azido nucleotide, or another suchnucleotide as is or may be known in the art, so as to not be capable ofbeing incorporated into an elongating nucleic acid chain during apolymerase reaction. In some instances, the nucleotide can include a3′-O-azido group, a 3′-O-azidomethyl group, a 3′-phosphorothioate group,a 3′-O-malonyl group, a 3′-O-alkyl hydroxylamino group, or a 3′-O-benzylgroup. In some instances, the nucleotide lacks a 3′ hydroxyl group.

The polymer can further have a binding or incorporation moiety in eachbranch or a subset of branches. Some examples of the binding orincorporation moiety include but are not limited to biotin, avidin,strepavidin or the like, polyhistidine domains, complementary pairednucleic acid domains, G-quartet forming nucleic acid domains,calmodulin, maltose-binding protein, cellulase, maltose, sucrose,glutathione-S-transferase, glutathione, O-6-methylguanine-DNAmethyltransferase, benzylguanine and derivatives thereof, benzylcysteineand derivatives thereof, an antibody, an epitope, a protein A, a proteinG. The binding or incorporation moiety can be any interactive moleculesor fragment thereof known in the art to bind to or facilitateinteractions between proteins, between proteins and ligands, betweenproteins and nucleic acids, between nucleic acids, or between smallmolecule interaction domains or moieties.

In some embodiments, a composition as provided herein may comprise oneor more elements of a complementary interaction moiety. Non-limitingexamples of complementary interaction moieties include, for example,biotin and avidin; SNAP-benzylguanosine; antibody or FAB and epitope;IgG FC and Protein A, Protein G, ProteinA/G, or Protein L; maltosebinding protein and maltose; lectin and cognate polysaccharide; ionchelation moieties, complementary nucleic acids, nucleic acids capableof forming triplex or triple helical interactions; nucleic acids capableof forming G-quartets, and the like. One of skill in the art willreadily recognize that many pairs of moieties exist and are commonlyused for their property of interacting strongly and specifically withone another; and thus any such complementary pair or set is consideredto be suitable for this purpose in constructing or envisioning thecompositions of the present disclosure. In some examples, a compositionas disclosed herein may comprise compositions in which one element of acomplementary interaction moiety is attached to one molecule ormultivalent ligand, and the other element of the complementaryinteraction moiety is attached to a separate molecule or multivalentligand. In some examples, a composition as disclosed herein may comprisecompositions in which both or all elements of a complementaryinteraction moiety are attached to a single molecule or multivalentligand. In some examples, a composition as disclosed herein may comprisecompositions in which both or all elements of a complementaryinteraction moiety are attached to separate arms of, or locations on, asingle molecule or multivalent ligand. In some examples, a compositionas disclosed herein may comprise compositions in which both or allelements of a complementary interaction moiety are attached to the samearm of, or locations on, a single molecule or multivalent ligand. Insome examples, compositions comprising one element of a complementaryinteraction moiety and compositions comprising another element of acomplementary interaction moiety may be simultaneously or sequentiallymixed. In some examples, interactions between molecules or particles asdisclosed herein allow for the association or aggregation of multiplemolecules or particles such that, for example, detectable signals areincreased. In some examples, fluorescent, colorimetric, or radioactivesignals are enhanced. In other examples, other interaction moieties asdisclosed herein, or as are known in the art, are contemplated. In someexamples, a composition as provided herein may be provided such that oneor more molecules comprising a first interaction moiety such as, forexample, one or more imidazole or pyridine moieties, and one or moreadditional molecules comprising a second interaction moiety such as, forexample, histidine residues, are simultaneously or sequentially mixed.In some examples, said composition comprises 1, 2, 3, 4, 5, 6, or moreimidazole or pyridine moieties. In some examples, said compositioncomprises 1, 2, 3, 4, 5, 6, or more histidine residues. In suchexamples, interaction between the molecules or particles as provided maybe facilitated by the presence of a divalent cation such as nickel,manganese, magnesium, calcium, strontium, or the like. For example, a(His)3 group may interact with a (His)3 group on another molecule orparticle via coordination of a nickel or manganese ion.

The multivalent binding or incorporation composition may comprise one ormore buffers, salts, ions, or additives. Representative additives mayinclude, but are not limited to, betaine, spermidine, detergents such asTriton X-100, Tween 20, SDS, or NP-40, ethylene glycol, polyethyleneglycol, dextran, polyvinyl alcohol, vinyl alcohol, methylcellulose,heparin, heparan sulfate, glycerol, sucrose, 1,2-propanediol, DMSO,N,N,N-trimethylglycine, ethanol, ethoxyethanol, propylene glycol,polypropylene glycol, block copolymers such as the Pluronic® seriespolymers, arginine, histidine, imidazole, or any combination thereof, orany substance known in the art as a DNA “relaxer” (i.e., a compound,with the effect of altering the persistence length of DNA, altering thenumber of within-polymer junctions or crossings, or altering theconformational dynamics of a DNA molecule such that the accessibility ofsites within the strand to DNA binding or incorporation moieties isincreased).

The multivalent binding or incorporation composition may includezwitterionic compounds as additives. Further representative additivesmay be found in Lorenz, T. C. J. Vis. Exp. (63), e3998, doi:10.3791/3998(2012), which is hereby incorporated by reference with respect to itsdisclosure of additives for the facilitation of nucleic acid binding ordynamics, or the facilitation of processes involving the manipulation,use, or storage of nucleic acids. In some instances, representativecations include, but are not limited to, sodium, magnesium, strontium,potassium, manganese, calcium, lithium, nickel, cobalt, or other suchcations as are known in the art to facilitate nucleic acid interactions,such as self-association, secondary or tertiary structure formation,base pairing, surface association, peptide association, protein binding,or the like.

Binding Between Target Nucleic Acid and Multivalent Binding orIncorporation Composition. When the multivalent binding or incorporationcomposition is used in replacement of a single unconjugated oruntethered nucleotide to form a complex with the polymerase and one ormore copies of the target nucleic acid, the local concentration of thenucleotide as well as the binding avidity of the complex (in cases wherea complex comprising two or more target nucleic acid molecules isformed) is increased many-fold, which in turn enhances the signalintensity, particularly the correct signal versus mismatch. The presentdisclosure contemplates contacting the multivalent binding orincorporation composition with a polymerase and a primed target nucleicacid to determine the formation of a ternary binding or incorporationcomplex.

FIG. 17 illustrates the use of the disclosed polymer-nucleotideconjugates for achieving increased signal intensity during binding,persistence, and washing/removal steps. Because of the increased localconcentration of the nucleotide on the polymer-nucleotide conjugateand/or the formation of non-covalent bonds with two or more primedtarget nucleic acid molecules, the binding between the polymerase, theprimed target strand, and the polymer-conjugated nucleotide, when thenucleotide is complementary to the next base of the target nucleic acid,becomes more favorable. The formed binding complex has a longerpersistence time, which in turn helps increase signal and shorten theimaging step. The high signal intensity resulting from the use of thedisclosed polymer nucleotide conjugates remains stable for the entirebinding and imaging steps. The strong binding between the polymerase,the primed target strand, and the polymer-conjugated nucleotide ornucleotide analog also means that the binding complex thus formed willremain stable during wash steps as other reaction mixture components andunmatched nucleotide analogs are washed away. After the imaging step,the binding complex can be destabilized (e.g., by changing the buffercomposition) and the primed target nucleic acid can then be extended forone base. After the extension, the binding and imaging steps can berepeated with the use of the disclosed polymer nucleotide conjugates todetermine the identity of the next base.

As an example, a graphical depiction of the increase in signal intensityduring binding, persistence, and washing/removal of a multivalentsubstrate as described herein is provided in FIG. 17, which isrepresentative of the changes in signal intensity that have beenobserved experimentally. Therefore, the compositions and methods of thepresent disclosure provide a robust and controllable means ofestablishing and maintaining a ternary enzyme complex, as well asproviding vastly improved means by which the presence of said complexmay be identified and/or measured, and a means by which the persistenceof said complex may be controlled. This provides important solutions toproblems such as that of determining the identity of the N+1 base innucleic acid sequencing applications.

Without intending to be bound by any particular theory, it has beenobserved that multivalent binding compositions disclosed hereinassociate with polymerase nucleotide complexes in order to form aternary binding complexes with a rate that is time-dependent, thoughsubstantially slower than the rate of association known to be obtainableby nucleotides in free solution. Thus, the on-rate (Kon) issubstantially and surprisingly slower than the on rate for singlenucleotides or nucleotides not attached to multivalent ligand complexes.Importantly, however, the off rate (Koff) of the multivalent ligandcomplex is substantially slower than that observed for nucleotides infree solution. Therefore, the multivalent ligand complexes of thepresent disclosure provide a surprising and beneficial improvement ofthe persistence of ternary polymerase-polynucleotide-nucleotidecomplexes (especially over such complexes that are formed with freenucleotides) allowing, for example, significant improvements in imagingquality for nucleic acid sequencing applications over currentlyavailable methods and reagents. Importantly, this property of themultivalent binding compositions disclosed herein renders the formationof visible ternary complexes controllable, such that subsequentvisualization, modification, or processing steps may be undertakenessentially without regard to the dissociation of the complex—that is,the complex can be formed, imaged, modified, or used in other ways asnecessary, and will remain stable until a user carries out anaffirmative dissociation step, such as exposing the complexes to adissociation buffer.

In some instances, the persistence times for the multivalent bindingcomplexes formed using the disclosed particle-nucleotide orpolymer-nucleotide conjugates may range from about 0.1 second to about600 second under non-destabilizing conditions. In some instances, thepersistence time may be at least 0.1 second, at least 1 second, at least2 seconds, at least 3 second, at least 4 second, at least 5 seconds, atleast 6 seconds, at least 7 seconds, at least 8 seconds, at least 9seconds, at least 10 seconds, at least 20 seconds, at least 30 second,at least 40 second, at least 50 seconds, at least 60 seconds, at least120 seconds, at least 180 seconds, at least 240 seconds, at least 300seconds, at least 360 seconds, at least 420 seconds, at least 480seconds, at least 540 seconds, or at least 600 seconds. In someinstances, the persistence time may range between any two of the valuesspecified in this paragraph. For example, in some instances, thepersistence time may range from about 10 seconds to about 360 seconds.Those of skill in the art will recognize that in some instances, thepersistence time may have any value within the range of values specifiedin this paragraph, e.g., 78 seconds.

In various examples, polymerases suitable for the binding orincorporation interaction describe herein include may include anypolymerase as is or may be known in the art. It is, for example, knownthat every organism encodes within its genome one or more DNApolymerases. Examples of suitable polymerases may include but are notlimited to: Klenow DNA polymerase, Thermus aquaticus DNA polymerase I(Taq polymerase), KlenTaq polymerase, and bacteriophage T7 DNApolymerase; human alpha, delta and epsilon DNA polymerases;bacteriophage polymerases such as T4, RB69 and phi29 bacteriophage DNApolymerases, Pyrococcus furiosus DNA polymerase (Pfu polymerase);Bacillus subtilis DNA polymerase III, and E. coli DNA polymerase IIIalpha and epsilon; 9 degree N polymerase, reverse transcriptases such asHIV type M or O reverse transcriptases, avian myeloblastosis virusreverse transcriptase, or Moloney Murine Leukemia Virus (MMLV) reversetranscriptase, or telomerase.

Further non-limiting examples of DNA polymerases can include those fromvarious Archaea genera, such as, Aeropyrum, Archaeglobus,Desulfurococcus, Pyrobaculum, Pyrococcus, Pyrolobus, Pyrodictium,Staphylothermus, Stetteria, Sulfolobus, Thermococcus, and Vulcanisaetaand the like or variants thereof, including such polymerases as areknown in the art such as Vent™, Deep Vent™, Pfu, KOD, Pfx, Therminator™,and Tgo polymerases. In some examples, the polymerase is a klenowpolymerase.

The ternary complex has longer persistence time when the nucleotide onthe polymer-nucleotide conjugate is complementary to the target nucleicacid than when it is non-complementary to the target nucleic acid. Theternary complex also has longer persistence time when the nucleotide onthe polymer-nucleotide conjugate is complementary to the target nucleicacid than a complementary nucleotide that is not conjugated or tethered.For example, in some embodiments, said ternary complexes may have apersistence time of less than 1s, greater than 1s, greater than 2s,greater than 3s, greater than 5s, greater than 10s, greater than 15s,greater than 20s, greater than 30s, greater than 60s, greater than 120s,greater than 360s, greater than 3600s, or more, or for a time lyingwithin a range defined by any two or more of these values.

The persistence time can be measured, for example, by observing theonset and/or duration of a binding complex, such as by observing asignal from a labeled component of the binding complex. For example, alabeled nucleotide or a labeled reagent comprising one or morenucleotides may be present in a binding complex, thus allowing thesignal from the label to be detected during the persistence time of thebinding complex.

It has been observed that different ranges of persistence times areachievable with different salts or ions, showing, for example, thatcomplexes formed in the presence of, for example, magnesium ions (Mg2+)form more quickly than complexes formed with other ions. It has alsobeen observed that complexes formed in the presence of, for example,strontium ions (Sr2+), form readily and dissociate completely or withsubstantial completeness upon withdrawal of the ion or upon washing withbuffer lacking one or more components of the present compositions, suchas, e.g., a polymer and/or one or more nucleotides, and/or one or moreinteraction moieties, or a buffer containing, for example, a chelatingagent which may cause or accelerate the removal of a divalent cationfrom the multivalent reagent containing complex. Thus, in some examples,a composition of the present disclosure comprises Mg2+. In someexamples, a composition of the present disclosure comprises Ca2+. Insome examples, a composition of the present disclosure comprises Sr2+.In some examples, a composition of the present disclosure comprisescobalt ions (Co2+). In some examples, a composition of the presentdisclosure comprises MgCl2. In some examples, a composition of thepresent disclosure comprises CaCl2. In some examples, a composition ofthe present disclosure comprises SrCl2. In some examples, a compositionof the present disclosure comprises CoCl2. In some examples, thecomposition comprises no, or substantially no magnesium. In someexamples, the composition comprises no, or substantially no calcium. Insome examples, the methods of the present disclosure provide for thecontacting of one or more nucleic acids with one or more of thecompositions disclosed herein, wherein said composition lacks either oneof calcium or magnesium or lacks both calcium or magnesium.

The dissociation of ternary complexes can be controlled by changing thebuffer conditions. After the imaging step, a buffer with increased saltcontent is used to cause dissociation of the ternary complexes such thatlabeled polymer-nucleotide conjugates can be washed out, providing ameans by which signals can be attenuated or terminated, such as in thetransition between one sequencing cycle and the next. This dissociationmay be affected, in some embodiments, by washing the complexes with abuffer lacking a necessary metal or cofactor. In some instances, a washbuffer may comprise one or more compositions for the purpose ofmaintaining pH control. In some instances, a wash buffer may compriseone or more monovalent cations, such as sodium. In some instances, awash buffer lacks or substantially lacks a divalent cation, for example,having no or substantially no strontium, calcium, magnesium, ormanganese. In some instances, a wash buffer further comprises achelating agent, such as, for example, EDTA, EGTA, nitrilotriaceticacid, polyhistidine, imidazole, or the like. In some instances, a washbuffer may maintain the pH of the environment at the same level as forthe bound complex. In some instances, a wash buffer may raise or lowerthe pH of the environment relative to the level seen for the boundcomplex. In some instances, the pH may be within a range from 2-4, 2-7,5-8, 7-9, 7-10, or lower than 2, or higher than 10, or a range definedby any two of the values provided herein.

Addition of a particular ion may affect the binding of the polymerase toa primed target nucleic acid, the formation of a ternary complex, thedissociation of a ternary complex, or the incorporation of one or morenucleotides into an elongating nucleic acid such as during a polymerasereaction. In some instances, relevant anions may comprise chloride,acetate, gluconate, sulfate, phosphate, or the like. In some instances,an ion may be incorporated into the compositions of the presentdisclosure by the addition of one or more acids, bases, or salts, suchas NiCl2, CoCl2, MgCl2, MnCl2, SrCl2, CaCl2, CaSO4, SrCO3, BaCl2 or thelike. Representative salts, ions, solutions and conditions may be foundin Remington: The Science and Practice of Pharmacy, 20th. Edition,Gennaro, A. R., Ed. (2000), which is hereby incorporated by reference inits entirety, and especially with respect to Chapter 17 and relateddisclosure of salts, ions, salt solutions, and ionic solutions.

The present disclosure contemplates contacting the multivalent bindingor incorporation composition comprising at least one particle-nucleotideconjugate with one or more polymerases. The contacting can be optionallydone in the presence of one or more target nucleic acids. In someexamples, said target nucleic acids are single stranded nucleic acids.In some examples, said target nucleic acids are primed single strandednucleic acids. In some examples, said target nucleic acids are doublestranded nucleic acids. In some examples, said contacting comprisescontacting the multivalent binding or incorporation composition with onepolymerase. In some examples, said contacting comprises the contactingof said composition comprising one or more nucleotides with multiplepolymerases. The polymerase can be bound to a single nucleic acidmolecule.

The binding between target nucleic acid and multivalent bindingcomposition may be provided in the presence of a polymerase that hasbeen rendered catalytically inactive. In one example, the polymerase mayhave been rendered catalytically inactive by mutation. In one example,the polymerase may have been rendered catalytically inactive by chemicalmodification. In some examples, the polymerase may have been renderedcatalytically inactive by the absence of a necessary substrate, ion, orcofactor. In some examples, the polymerase enzyme may have been renderedcatalytically inactive by the absence of magnesium ions.

The binding between a target nucleic acid and multivalent bindingcomposition described herein may occur in the presence of a polymerasewherein the binding solution, reaction solution, or buffer lacksmagnesium or manganese. In another example, the binding between thetarget nucleic acid and multivalent binding composition occurs in thepresence of a polymerase wherein the binding solution, reactionsolution, or buffer comprises calcium or strontium.

In some instances, when catalytically inactive polymerases are used tohelp a nucleic acid interact with a multivalent binding composition, theinteraction between said composition and said polymerase stabilizes aternary complex so as to render the complex detectable by fluorescenceor by other methods as disclosed herein or otherwise known in the art.Unbound polymer-nucleotide conjugates may optionally be washed awayprior to detection of the ternary binding complex.

Contacting of one or more nucleic acids with the polymer-nucleotideconjugates disclosed herein may occur in a solution containing eitherone of calcium or magnesium or containing both calcium and magnesium. Inanother example, the contacting of one or more nucleic acids with thepolymer-nucleotide conjugates disclosed herein occurs in a solutionlacking either one of calcium or magnesium, or lacking both calcium ormagnesium, and in a separate step, without regard to the order of thesteps, one of calcium or magnesium, or both calcium and magnesium, maybe added to the solution. In some embodiments, the contacting of one ormore nucleic acids with the polymer-nucleotide conjugates disclosedherein occurs in a solution lacking strontium, and comprises in aseparate step, without regard to the order of the steps, adding to thesolution strontium.

Illustrative Embodiment 1

The disclosed methods of determining the sequence of a target nucleicacid comprise: a) contacting a double-stranded or partiallydouble-stranded target nucleic acid molecule comprising the templatestrand to be sequenced and a primer strand to be elongated with one ormore of the disclosed nucleic acid binding compositions; and b)detecting the binding of a nucleic acid binding composition to thenucleic acid molecule, thereby determining the presence of one of saidone or more nucleic acid binding compositions on said nucleic acidmolecule and the identity of the next nucleotide (i.e., the N+1 orterminal nucleotide) to be incorporated into the complementary strand.

The sequencing method may further comprise incorporating the N+1 orterminal nucleotide into the primer strand, and then repeating thecontacting, detecting, and incorporating steps for one or moreadditional iterations, thereby determining the sequence of the templatestrand of the nucleic acid molecule. After the step of detecting theternary binding complex, the primed strand of the primed target nucleicacid is extended for one base before another round of analysis isperformed. The primed target nucleic acid can be extended using theconjugated nucleotide that is attached to the polymer in the multivalentbinding composition or using an unconjugated or untethered freenucleotide that is provided after the multivalent binding compositionhas been removed.

The extension of the primed target nucleic acid may be prevented orinhibited due to a blocked nucleotide on the strand or the use ofpolymerase that is catalytically inactive. When the nucleotide in thepolymer-nucleotide conjugate has a blocking group that prevents theextension of the nucleic acid, incorporation of a nucleotide may beachieved by the removal of a blocking group from said nucleotide (suchas by detachment of said nucleotide from its polymer, branched polymer,dendrimer, particle, or the like). When the extension of the primedtarget nucleic acid is inhibited due to the use of polymerase that iscatalytically inactive, incorporation of a nucleotide may be achieved bythe provision of a cofactor or activator such as a metal ion.

Detection of the ternary complex is achieved prior to, concurrentlywith, or following the incorporation of the nucleotide residue. In someinstances, a primed target nucleic acid may comprise a target nucleicacid with multiple primed locations for the attachment of polymerasesand/or nucleic acid binding moieties. In some instances, multiplepolymerases may be attached to a single target nucleic acid molecule,such as at multiple sites within a target nucleic acid molecule. In someinstances, multiple polymerases may be bound to a multivalent bindingcomposition disclosed herein comprising multiple nucleotides. In someinstances, a target nucleic acid molecule may be a product of a stranddisplacement synthesis, a rolling circle amplification, a concatenationor fusion of multiple copies of a query sequence, or other such methodsas are known in the art or as are disclosed elsewhere herein to producenucleic acid molecules comprising multiple copies of an identicalsequence. Therefore, in some instances, multiple polymerases may beattached at multiple identical or substantially identical locationswithin a target nucleic acid, which comprises multiple identical orsubstantially identical copies of a query sequence. In some instances,said multiple polymerases may then be involved in interactions with oneor more multivalent binding complexes; however, in some examples, thenumber of binding sites within a target nucleic acid is at least two,and the number of nucleotides or substrate moieties present on aparticle-nucleotide conjugate such as a polymer-nucleotide conjugate isalso greater than or equal to two.

In some examples, the multivalent binding compositions are provided incombination with other elements such as to provide optimized signals,for example to provide identification of a nucleotide at a particularposition in a nucleic acid sequence. In some instances, the compositionsdisclosed herein are provided in combination with a surface providinglow background binding or low levels of protein binding, such as, forexample, a hydrophilic or polymer coated surface. Representativesurfaces may be found, for example, in U.S. patent application Ser. No.16/363,842, the contents of which are hereby incorporated by referencein their entirety.

In some instances, the nucleic acid molecule is tethered to the surfaceof a solid support, e.g., through hybridization of the template strandto an adapter nucleic acid sequence or primer nucleic acid sequence thatis tethered to the solid support. In some instances, the solid supportcomprises a glass, fused-silica, silicon, or polymer substrate. In someinstances, the solid support comprises a low non-specific bindingcoating comprising one or more hydrophilic polymer layers (e.g., PEGlayers) where at least one of the hydrophilic polymer layers comprises abranched polymer molecule (e.g., a branched PEG molecule comprising 4,8, 16, or 32 branches).

The solid support comprises oligonucleotide adapters or primers tetheredto at least one hydrophilic polymer layer at a surface density rangingfrom about 1,000 primer molecules per μm2 to about 1,000,000 primermolecules per μm². In some instances, the surface density ofoligonucleotide primers may be at least 1,000, at least 10,000, at least100,000, or at least 1,000,000 molecules per μm². In some instances, thesurface density of oligonucleotide primers may be at most 1,000,000, atmost 100,000, at most 10,000, or at most 1,000 molecules per μm². Any ofthe lower and upper values described in this paragraph may be combinedto form a range included within the present disclosure, for example, insome instances the surface density of primers may range from about10,000 molecules per μm² to about 100,000 molecules per μm². Those ofskill in the art will recognize that the surface density of primermolecules may have any value within this range, e.g., about 455,000molecules per μm².

One of ordinary skill would recognize that in a series of iterativesequencing reactions, occasionally one or more sites will fail toincorporate a nucleotide during a given cycle, thus leading one or moresites to be unsynchronized with the bulk of the elongating nucleic acidchains. Under conditions in which sequencing signals are derived fromreactions occurring on single copies of a target nucleic acid, thesefailures to incorporate will yield discrete errors in the outputsequence. It is an object of the present disclosure to describe methodsfor reducing this type of error in sequencing reactions. For example,the use of multivalent substrates that are capable of incorporation intothe elongating strand, by providing increased probabilities of rebindingupon premature dissociation of a ternary polymerase complex, can reducethe frequency of “skipped” cycles in which a base is not incorporated.Thus, in some examples, the present disclosure contemplates the use ofmultivalent substrates as disclosed herein in which the nucleosidemoiety is comprised within a nucleotide having a free, or reversiblymodified, 5′ phosphate, diphosphate, or triphosphate moiety, and whereinthe nucleotide is connected to the particle or polymer as disclosedherein, through a labile or cleavable linkage. In some examples, thepresent disclosure contemplates a reduction in the intrinsic error ratedue to skipped incorporations as a result of the use of the multivalentsubstrates disclosed herein.

The present disclosure also contemplates sequencing reactions in whichsequencing signals from or relating to a given sequence are derived fromor originate within definable regions containing multiple copies of thetarget sequence. Sequencing methods incorporating multiple copies of atarget sequence are advantageous in that signals can be amplified due tothe presence of multiple simultaneous sequencing reactions within thedefined region, each providing its own signal. The presence of multiplesignals within a defined area also reduces the impact of any singleskipped cycle, due to the fact that the signal from a large number ofcorrect base calls can overwhelm the signal from a smaller number ofskipped or incorrect base calls. The present disclosure furthercontemplates the inclusion of free, unlabeled nucleotides duringelongation reactions, or during a separate part of the elongation cycle,in order to provide incorporation at sites that may have been skipped inprevious cycles. For example, during or following an incorporationcycle, unlabeled blocked nucleotides may be added such that they may beincorporated at skipped sites. The unlabeled blocked nucleotides may beof the same type or types as the nucleotide attached to the multivalentbinding substrate or substrates that are or were present during aparticular cycle, or a mixture of 1, 2, 3, 4 or more types of unlabeledblocked nucleotides may be included.

When each sequencing cycle proceeds perfectly, each reaction within thedefined region will provide an identical signal. However, as notedelsewhere herein, in a series of iterative sequencing reactions,occasionally one or more sites will fail to incorporate a nucleotideduring a given cycle, thus leading one or more sites to beunsynchronized with the bulk of the elongating nucleic acid chains. Thisissue, referred to as “phasing,” leads to degradation of the sequencingsignal as the signal is contaminated with spurious signals from siteshaving skipped one or more cycles. This, in turn, creates the potentialfor errors in base identification. The progressive accumulation ofskipped cycles through multiple cycles also reduces the effective readlength, due to progressive degradation of the sequencing signal witheach cycle. It is a further object of this disclosure to provide methodsfor reducing phasing errors and/or to improve read length in sequencingreactions.

The sequencing method can include contacting a target nucleic acid ormultiple target nucleic acids, comprising multiple linked or unlinkedcopies of a target sequence, with the multivalent binding compositionsdescribed herein. Contacting said target nucleic acid, or multipletarget nucleic acids comprising multiple linked or unlinked copies of atarget sequence, with one or more particle-nucleotide conjugates mayprovide a substantially increased local concentration of the correctnucleotide being interrogated in a given sequencing cycle, thussuppressing signals from improper incorporations or phased nucleic acidchains (i.e., those elongating nucleic acid chains which have had one ormore skipped cycles).

Methods of obtaining nucleic acid sequence information can includecontacting a target nucleic acid, or multiple target nucleic acids,wherein said target nucleic acid or multiple target nucleic acidscomprise multiple linked or unlinked copies of a target sequence, withone or more particle-nucleotide conjugates. This method results in areduction in the error rate of sequencing as indicated by reduction inthe misidentification of bases, the reporting of nonexistent bases, orthe failure to report correct bases. In some embodiments, said reductionin the error rate of sequencing may comprise a reduction of 5%, 10%,15%, 20% 25%, 50%, 75%, 100%, 150%, 200%, or more compared to the errorrate observed using monovalent ligands, including free nucleotides,labeled free nucleotides, protein or peptide bound nucleotides, orlabeled protein or peptide bound nucleotides.

The method of obtaining nucleic acid sequence information can includecontacting a target nucleic acid, or multiple target nucleic acids,wherein said templet nucleic acid or multiple target nucleic acidscomprise multiple linked or unlinked copies of a target sequence, withone or more particle-nucleotide conjugates. This method results in anincrease in average read length of 5%, 10%, 15%, 20%, 25%, 50%, 75%,100%, 150%, 200%, 300%, or more compared to the average read lengthobserved using monovalent ligands, including free nucleotides, labeledfree nucleotides, protein or peptide bound nucleotides, or labeledprotein or peptide bound nucleotides.

Disclosed herein are methods of obtaining nucleic acid sequenceinformation, said methods comprising contacting a target nucleic acid,or multiple target nucleic acids, wherein said target nucleic acid ormultiple target nucleic acids comprise multiple linked or unlinkedcopies of a target sequence, with one or more particle-nucleotideconjugates. Such methods may result in an increase in average readlength of 10 nucleotides (NT), 20 NT, 25 NT, 30 NT, 50 NT, 75 NT, 100NT, 125 NT, 150 NT, 200 NT, 250 NT, 300 NT, 350 NT, 400 NT, 500 NT, ormore compared to the average read length observed using monovalentligands, including free nucleotides, labeled free nucleotides, proteinor peptide bound nucleotides, or labeled protein or peptide boundnucleotides.

In some instances, the disclosed compositions and methods may result inaverage read lengths for sequencing applications that range from 100nucleotides to 1,000 nucleotides. In some instances, the average readlength may be at least 100 nucleotides, at least 200 nucleotides, atleast 225 nucleotides, at least 250 nucleotides, at least 275nucleotides, at least 300 nucleotides, at least 325 nucleotides, atleast 350 nucleotides, at least 375 nucleotides, at least 400nucleotides, at least 425 nucleotides, at least 450 nucleotides, atleast 475 nucleotides, at least 500 nucleotides, at least 525nucleotides, at least 550 nucleotides, at least 575 nucleotides, atleast 600 nucleotides, at least 625 nucleotides, at least 650nucleotides, at least 675 nucleotides, at least 700 nucleotides, atleast 725 nucleotides, at least 750 nucleotides, at least 775nucleotides, at least 800 nucleotides, at least 825 nucleotides, atleast 850 nucleotides, at least 875 nucleotides, at least 900nucleotides, at least 925 nucleotides, at least 950 nucleotides, atleast 975 nucleotides, or at least 1,000 nucleotides. In some instances,the average read length may be a range bounded by any two of the valueswithin this range, e.g., an average read length ranging from 375nucleotides to 825 nucleotides. Those of skill in the art will recognizethat in some instances, the average read length may have any valuewithin the range specified in this paragraph, e.g., 523 nucleotides.

In some instances, the use of multivalent binding compositions describedherein for sequencing effectively shortens the sequencing time. Thesequencing reaction cycle comprising the contacting, detecting, andincorporating steps is performed in a total time ranging from about 5minutes to about 60 minutes. In some instances, the sequencing reactioncycle is performed in at least 5 minutes, at least 10 minutes, at least20 minutes, at least 30 minutes, at least 40 minutes, at least 50minutes, or at least 60 minutes. In some instances, the sequencingreaction cycle is performed in at most 60 minutes, at most 50 minutes,at most 40 minutes, at most 30 minutes, at most 20 minutes, at most 10minutes, or at most 5 minutes. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances thesequencing reaction cycle may be performed in a total time ranging fromabout 10 minutes to about 30 minutes. Those of skill in the art willrecognize that the sequencing cycle time may have any value within thisrange, e.g., about 16 minutes.

In some instances, the disclosed compositions and methods for nucleicacid sequencing will provide an average base-calling accuracy of atleast 80%, at least 85%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 98%, at least 99%, at least 99.5%, at least 99.8%,or at least 99.9% correct over the course of a sequencing run. In someinstances, the disclosed compositions and methods for nucleic acidsequencing will provide an average base-calling accuracy of at least80%, at least 85%, at least 90%, at least 92%, at least 94%, at least96%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or atleast 99.9% correct per every 1,000 bases, 10,0000 bases, 25,000 bases,50,000 bases, 75,000 bases, or 100,000 bases called.

In some instances, the use of multivalent binding compositions disclosedherein for sequencing provides more accurate base readout. In someinstances, the disclosed compositions and methods for nucleic acidsequencing will provide an average Q-score for base-calling accuracyover a sequencing run that ranges from about 20 to about 50. In someinstances, the average Q-score is at least 20, at least 25, at least 30,at least 35, at least 40, at least 45, or at least 50. Those of skill inthe art will recognize that the average Q-score may have any valuewithin this range, e.g., about 32.

In some instances, the disclosed compositions and methods for nucleicacid sequencing will provide a Q-score of greater than 30 for at least50%, at least 60%, at least 70%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% of the terminal (orN+1) nucleotides identified. In some instances, the disclosedcompositions and methods for nucleic acid sequencing will provide aQ-score of greater than 35 for at least 50%, at least 60%, at least 70%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% of the terminal (or N+1) nucleotides identified. In someinstances, the disclosed compositions and methods for nucleic acidsequencing will provide a Q-score of greater than 40 for at least 50%,at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99% of the terminal (or N+1)nucleotides identified. In some instances, the disclosed compositionsand methods for nucleic acid sequencing will provide a Q-score ofgreater than 45 for at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% of the terminal (or N+1) nucleotides identified. In some instances,the disclosed compositions and methods for nucleic acid sequencing willprovide a Q-score of greater than 50 for at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% of the terminal (or N+1) nucleotidesidentified.

The disclosed low non-specific binding supports and associated nucleicacid hybridization and amplification methods may be used for theanalysis of nucleic acid molecules derived from any of a variety ofdifferent cell, tissue, or sample types known to those of skill in theart. For example, nucleic acids may be extracted from cells, or tissuesamples comprising one or more types of cells, derived from eukaryotes(such as animals, plants, fungi, or protista), archaebacteria, oreubacteria. In some instances, nucleic acids may be extracted fromprokaryotic or eukaryotic cells, such as adherent or non-adherenteukaryotic cells. Nucleic acids are variously extracted from, forexample, primary or immortalized rodent, porcine, feline, canine,bovine, equine, primate, or human cell lines. Nucleic acids may beextracted from any of a variety of different cell, organ, or tissuetypes (e.g., white blood cells, red blood cells, platelets, epithelialcells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts,skeletal muscle cells, smooth muscle cells, gametes, or cells from theheart, lungs, brain, liver, kidney, spleen, pancreas, thymus, bladder,stomach, colon, or small intestine). Nucleic acids may be extracted fromnormal or healthy cells. In another example, or in combination, nucleicacids are extracted from diseased cells, such as cancerous cells, orfrom pathogenic cells that are infecting a host. Some nucleic acids maybe extracted from a distinct subset of cell types, e.g., immune cells(such as T cells, cytotoxic (killer) T cells, helper T cells, alpha betaT cells, gamma delta T cells, T cell progenitors, B cells, B-cellprogenitors, lymphoid stem cells, myeloid progenitor cells, lymphocytes,granulocytes, Natural Killer cells, plasma cells, memory cells,neutrophils, eosinophils, basophils, mast cells, monocytes, dendriticcells, and/or macrophages, or any combination thereof), undifferentiatedhuman stem cells, human stem cells that have been induced todifferentiate, rare cells (e.g., circulating tumor cells (CTCs),circulating epithelial cells, circulating endothelial cells, circulatingendometrial cells, bone marrow cells, progenitor cells, foam cells,mesenchymal cells, or trophoblasts). Nucleic acids may further comprisenucleic acids derived from viral samples and from subviral pathogens,such as viroids and infectious RNAs. Nucleic acids may be derived fromclinical or other samples, such as sputum, saliva, ocular fluid,synovial fluid, blood, feces, urine, tissue exudate, sweat, pus,drainage fluid or the like. Nucleic acids may further be derived fromplant or fungal samples, such as leaf, cambium, root, meristem, pollen,ovum, seed, spore, inflorescence, mycelium, or the like. Nucleic acidsmay also be derived from environmental or industrial samples, such aswater, air, dust, food, or the like. Other cells, tissues, and samplesare contemplated and consistent with the disclosure herein.

Nucleic acid extraction from cells or other biological samples may beperformed using any of a number of techniques known to those of skill inthe art. For example, a DNA extraction procedure may comprise (i)collection of the cell sample or tissue sample from which DNA is to beextracted, (ii) disruption of cell membranes (i.e., cell lysis) torelease DNA and other cytoplasmic components, (iii) treatment of thelysed sample with a concentrated salt solution to precipitate proteins,lipids, and RNA, followed by centrifugation to separate out theprecipitated proteins, lipids, and RNA, and (iv) purification of DNAfrom the supernatant to remove detergents, proteins, salts, or otherreagents used during the cell membrane lysis step.

A variety of suitable commercial nucleic acid extraction andpurification kits are consistent with the disclosure herein. Examplesinclude, but are not limited to, the QIAamp kits (for isolation ofgenomic DNA from human samples) and DNAeasy kits (for isolation ofgenomic DNA from animal or plant samples) from Qiagen (Germantown, Md.),or the Maxwell® and ReliaPrep™ series of kits from Promega (Madison,Wis.).

Illustrative Embodiment 2

Provided herein are methods for attaching a target nucleic acid moleculeto a surface, the methods comprising: bringing a mixture comprising saidtarget nucleic acid molecule at a concentration of 1 nanomolar or lessin contact with a hydrophilic surface comprising a capture probe coupledthereto under conditions sufficient for said target nucleic acidmolecule to be captured by said capture probe in a time period of lessthan 30 minutes.

In some instances, said mixture comprises a polar aprotic solvent. Insome instances, the polar aprotic solvent comprises formamide. In someinstances, said capture probe is a nucleic acid molecule. In someinstances, said concentration is 0.50 nanomolar or less. In someinstances, said concentration is 250 picomolar or less. In someinstances, said concentration is 100 picomolar or less. In someinstances, said time period is less than or equal to 20 minutes. In someinstances, said time period is less than or equal to 15 minutes. In someinstances, said time period is less than or equal to 10 minutes. In someinstances, said time period is less than or equal to 5 minutes.

In some instances, said hydrophilic surface is maintained at atemperature of about 30 degrees Celsius to about 70 degrees Celsius. Insome instances, said hydrophilic surface is maintained at asubstantially constant temperature. In some instances, methods furthercomprise hybridizing the target nucleic acid molecule to the captureprobe at a hybridization efficiency that is increased as compared to acomparable hybridization reaction performed for 120 minutes at 90degrees Celsius for 5 minutes followed by cooling for 120 minutes toreach a final temperature of 37 degrees Celsius in a buffer compositioncomprising saline-sodium citrate. In some instances, methods furthercomprise hybridizing the target nucleic acid molecule to the captureprobe with a hybridization stringency of at least 80%.

In some instances, the hydrophilic surface exhibits a level ofnon-specific Cyanine 3 dye absorption of less than about 0.25 moleculesper square micrometer. In some instances, the mixture further comprisesa pH buffer comprising 2-(N-morpholino)ethanesulfonic acid,acetonitrile, 3-(N-morpholino)propanesulfonic acid, methanol, or acombination thereof. In some instances, the mixture further comprises acrowding agent selected from the group consisting of polyethyleneglycol, dextran, hydroxypropyl methyl cellulose, hydroxyethyl methylcellulose, hydroxybutyl methyl cellulose, hydroxypropyl cellulose,methyl cellulose, and hydroxyl methyl cellulose, and any combinationthereof. In some instances, the hydrophilic surface comprises one ormore hydrophilic polymer layers. In some instances, the one or morehydrophilic polymer layers comprises a molecule selected from the groupconsisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA),poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid)(PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methylmethacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, anddextran. In some instances, the one or more hydrophilic polymer layerscomprises at least one dendrimer.

Provided herein are methods for hybridizing a target nucleic acidmolecule to a nucleic acid molecule coupled to a hydrophilic polymersurface, the method comprising: (a) providing at least one nucleic acidmolecule that is coupled to a hydrophilic polymer surface; and (b)bringing the at least one nucleic acid molecule coupled to the polymersurface into contact with a hybridizing composition comprising a targetnucleic acid molecule at a concentration of 1 nanomolar or less underconditions sufficient for said target nucleic acid molecule to hybridizeto the at least one nucleic acid molecule coupled to the polymer surfacein 30 minutes or less. In some instances, said conditions are maintainedat a substantially constant temperature.

In some instances, the hydrophilic polymer surface has a water contactangle of less than 45 degrees. In some instances, the target nucleicacid molecule is present in the hybridizing composition at aconcentration of 0.50 nanomolar or less. In some instances, the targetnucleic acid molecule is present in the hybridizing composition at aconcentration of 250 picomolar or less. In some instances, the targetnucleic acid molecule is present in the hybridizing composition at aconcentration of 100 picomolar or less. In some instances, bringing theat least one nucleic acid molecule coupled to the polymer surface intocontact with the hybridization composition is performed for a timeperiod of less than 30 minutes. In some instances, the time period isless than 20 minutes. In some instances, the time period is less than 15minutes. In some instances, the time period is less than 10 minutes. Insome instances, the time period is less than 5 minutes.

In some instances, methods further comprise hybridizing the targetnucleic acid molecule to the at least one nucleic molecule coupled tothe polymer surface at a hybridization efficiency that is increased ascompared to a comparable hybridization reaction performed for 120minutes at 90 degrees Celsius for 5 minutes followed by cooling for 120minutes to reach a final temperature of 37 degrees Celsius in a buffercomprising saline-sodium citrate. In some instances, the temperature isfrom about 30 degrees Celsius to 70 degrees Celsius. In some instances,the temperature is about 50 degrees Celsius. In some instances, methodsfurther comprise hybridizing the target nucleic acid molecule to the atleast one nucleic acid molecule with a hybridization stringency of atleast 80%. In some instances, the hydrophilic polymer surface exhibits alevel of non-specific Cyanine 3 dye absorption of less than about 0.25molecules per square micrometer.

In some instances, the hybridization composition further comprises: (a)at least one organic solvent having a dielectric constant of no greaterthan about 115 as measured at 68 degrees Fahrenheit; and (b) a pHbuffer. In some instances, the hybridization composition furthercomprises: (a) at least one organic solvent that is polar and aprotic;and (b) a pH buffer. In some instances, the at least one organic solventcomprises at least one functional group selected from hydroxy, nitrile,lactone, sulfone, sulfite, and carbonate. In some instances, the atleast one organic solvent comprises formamide. In some instances, the atleast one organic solvent is miscible with water. In some instances, theat least one organic solvent is at least about 5% by volume based on thetotal volume of the hybridizing composition. In some instances, the atleast one organic solvent is at most about 95% by volume based on thetotal volume of the hybridizing composition.

In some instances, the pH buffer is at most about 90% by volume of thetotal volume of the hybridizing composition. In some instances, the pHbuffer comprises 2-(N-morpholino)ethanesulfonic acid, acetonitrile,3-(N-morpholino)propanesulfonic acid, methanol, or a combinationthereof. In some instances, the pH buffer further comprises a secondorganic solvent. In some instances, the pH buffer is present in thehybridizing composition in an amount that is effective to maintain thepH of the hybridizing composition in a range of about 3 to about 10.

In some instances, the hybridizing composition further comprises amolecular crowding agent. In some instances, the molecular crowdingagent is selected from the group consisting of polyethylene glycol,dextran, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose,hydroxybutyl methyl cellulose, hydroxypropyl cellulose, methylcellulose, and hydroxyl methyl cellulose, and any combination thereof.In some instances, the molecular crowding agent is polyethylene glycol.In some instances, the molecular crowding agent has a molecular weightin the range of about 5,000 to 40,000 Daltons. In some instances, anamount of the molecular crowding agent is at least about 5% by volumebased on the total volume of the hybridizing composition. In someinstances, an amount of the molecular crowding agent is at most about50% by volume based on the total volume of the hybridizing composition.In some instances, the at least one nucleic acid molecule coupled to thepolymer surface is coupled to the polymer surface through covalentbonding.

In some instances, the hydrophilic polymer surface comprises one or morehydrophilic polymer layers, and the at least one nucleic acid moleculeis coupled to the one or more hydrophilic polymer layers. In someinstances, the one or more hydrophilic polymer layers comprises amolecule selected from the group consisting of polyethylene glycol(PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA),poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA),poly-lysine, poly-glucoside, streptavidin, and dextran. In someinstances, the one or more hydrophilic polymer layers comprises at leastone dendrimer.

Provided herein are methods of attaching a target nucleic acid to asurface, comprising: (a) providing at least one surface bound nucleicacid that is attached to a polymer surface having a water contact angleof less than 45 degrees; and (b) bringing the surface bound nucleic acidinto contact with a hybridizing composition under isothermal conditions,wherein the hybridizing composition comprises: (i) the target nucleicacid; (ii) at least one organic solvent having a dielectric constant ofno greater than about 115 when measured at 68 degrees Fahrenheit; and(iii) a pH buffer.

In some instances, the organic solvent is a polar aprotic solvent. Insome instances, the organic solvent is an organic solvent having adielectric constant of no greater than 40 when measured at 68 degreesFahrenheit. In some instances, the organic solvent is acetonitrile,alcohol, or formamide. In some instances, the organic solvent comprisesat least one functionality selected from hydroxy, nitrile, lactone,sulfone, sulfite, and carbonate. In some instances, the organic solventis miscible with water. In some instances, the organic solvent ispresent in an amount effective to denature a double stranded nucleicacid. In some instances, an amount of the organic solvent is at leastabout 5% by volume based on the total volume of the hybridizingcomposition. In some instances, an amount of the organic solvent is inthe range of about 5% to 95% by volume based on the total volume of thehybridizing composition. In some instances, an amount of the pH bufferis no greater than 90% by volume based on the total volume of thehybridizing composition. In some instances, the hybridizing compositionfurther comprises a molecular crowding agent. In some instances, themolecular crowding agent is selected from the group consisting ofpolyethylene glycol (PEG), dextran, hydroxypropyl methyl cellulose(HPMC), hydroxyethyl methyl cellulose (HEMC), hydroxybutyl methylcellulose, hydroxypropyl cellulose, methycellulose, and hydroxyl methylcellulose, and any combination thereof. In some instances, the molecularcrowding agent is polyethylene glycol (PEG). In some instances, themolecular crowding agent has a molecular weight in the range of about5,000 to 40,000 Daltons. In some instances, an amount of the molecularcrowding agent is at least about 5% by volume based on the total volumeof the hybridizing composition. In some instances, an amount of themolecular crowding agent is less than 50% by volume based on the totalvolume of the hybridizing composition. In some instances, methodsfurther comprise an additive for controlling a melting temperature ofthe target nucleic acid. In some instances, an amount of the additivefor controlling melting temperature of the target nucleic acid is atleast about 2% by volume based on the total volume of the hybridizingcomposition. In some instances, an amount of the additive forcontrolling melting temperature of the nucleic acid is in the range ofabout 2% to 50% by volume based on the total volume of the hybridizingcomposition. In some instances, the pH buffer comprises at least onebuffering agent selected from the group consisting of Tris, HEPES (e.g.,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TAPS (e.g.,[tris(hydroxymethyl)methylamino]propanesulfonic acid), Tricine, Bicine,Bis-Tris, sodium hydroxide (NaOH), potassium hydroxide (KOH), TES (e.g.,2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonicacid), EPPS (e.g., 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid,4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid,N-(2-Hydroxyethyl)piperazine-N-(3-propanesulfonic acid)), and MOPS(e.g., 3-(N-morpholino)propanesulfonic acid). In some instances, the pHbuffer further comprises a second organic solvent. In some instances,the pH buffer comprises MOPS and methanol. In some instances, an amountof the pH buffer is effective to maintain the pH of the hybridizingcomposition to be in the range of about 3 to about 10.

In some instances, the surface bound nucleic acid is coupled to thesurface through covalent or noncovalent bonding. In some instances, thepolymer surface comprises one or more hydrophilic polymer layers, andthe surface bound nucleic acid is coupled to the one or more hydrophilicpolymer layers. In some instances, no more than 10% of the targetnucleic acid is associated with the surface without hybridizing to thepolymer surface bound nucleic acid. In some instances, the polymersurface exhibits a level of non-specific cyanine 3 (Cy3) dye absorptionof less than about 0.25 molecules per micrometer squared (μm²). In someinstances, the one or more hydrophilic polymer layers comprises amolecule selected from the group consisting of polyethylene glycol(PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA),poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA),poly-lysine, poly-glucoside, streptavidin, and dextran. In someinstances, the one or more hydrophilic polymer layers comprise at leastone dendrimer.

In some instances, bringing the surface bound nucleic acid into contactwith the hybridizing composition is performed for a period of no morethan 25 minutes. In some instances, bringing the surface bound nucleicacid into contact with the hybridizing composition is performed for aperiod of no more than 15 minutes. In some instances, bringing thesurface bound nucleic acid into contact with the hybridizing compositionis performed for a period between 2-25 minutes. In some instances, theisothermal conditions are at a temperature in the range of about 30 to70 degrees Celsius. In some instances, attaching the target nucleic acidmolecule to the surface comprises hybridizing the target nucleic acid tothe surface bound nucleic acid with a hybridization stringency of atleast 80%. In some instances, attaching the target nucleic acid moleculeto the surface comprises hybridizing the target nucleic acid to thesurface bound nucleic with an increased hybridization efficiency, ascompared to a comparable hybridization reaction wherein the organicbuffer is a saline-sodium citrate and hybridizing is performed for 120minutes at 90 degrees Celsius for 5 minutes followed by cooling for 120minutes to reach a final temperature of 37 degrees Celsius. In someinstances, the target nucleic acid is present in the hybridizingcomposition at a 1 nanomolar concentration or less. In some instances,the target nucleic acid is present in the hybridizing composition at a250 picomolar concentration or less. In some instances, the targetnucleic acid is present in the hybridizing composition at a 100picomolar concentration or less. In some instances, the target nucleicacid is present in the hybridizing composition at a 50 picomolarconcentration or less. In some instances, methods further comprisehybridizing at least a portion of the surface bound nucleic acid to atleast a portion of the target nucleic acid in the hybridizingcomposition, wherein hybridizing does not consist of cooling.

Provided herein are methods of hybridization, the methods comprising:(a) providing at least one surface bound nucleic acid molecule coupledto a surface; and (b) bringing the at least one surface bound nucleicacid molecule into contact with a hybridizing composition comprising atarget nucleic acid molecule, wherein the hybridizing compositioncomprises: (i) at least one organic solvent; and (ii) a pH buffer. Insome instances, the surface exhibits a level of non-specific Cy3 dyeabsorption corresponding to less than about 0.25 molecules/μm² whenmeasured by a fluorescence imaging system under non-signal saturatingconditions. In some instances, no more than 5% of a total number of thetarget nucleic acid molecule is associated with the surface withouthybridizing to the surface bound nucleic acid molecule.

In some instances, the surface bound nucleic acid molecule is coupled tothe surface by being tethered to the surface. In some instances, thesurface is a hydrophilic polymer surface. In some instances, the surfacehas a water contact angle of less than 45 degrees. In some instances,the at least one organic solvent has a dielectric constant of no greaterthan about 115 when measured at 68 degrees Fahrenheit. In someinstances, the organic solvent is a polar aprotic solvent. In someinstances, the organic solvent is an organic solvent having a dielectricconstant of no greater than 40 when measured at 68 degrees Fahrenheit.In some instances, the organic solvent is acetonitrile, alcohol, orformamide. In some instances, the organic solvent comprises at least onefunctionality selected from hydroxy, nitrile, lactone, sulfone, sulfite,and carbonate. In some instances, the organic solvent is miscible withwater. In some instances, the organic solvent is present in an amounteffective to denature a double stranded nucleic acid. In some instances,an amount of the organic solvent is at least about 5% by volume based onthe total volume of the hybridizing composition. In some instances, anamount of the organic solvent is in the range of about 5% to 95% byvolume based on the total volume of the hybridizing composition. In someinstances, an amount of the pH buffer is no greater than 90% by volumebased on the total volume of the hybridizing composition. In someinstances, the hybridizing composition further comprises a molecularcrowding agent. In some instances, the molecular crowding agent isselected from the group consisting of polyethylene glycol (PEG),dextran, hydroxypropyl methyl cellulose (HPMC), hydroxyethyl methylcellulose (HEMC), hydroxybutyl methyl cellulose, hydroxypropylcellulose, methycellulose, and hydroxyl methyl cellulose, and anycombination thereof. In some instances, the molecular crowding agent ispolyethylene glycol (PEG). In some instances, the molecular crowdingagent has a molecular weight in the range of about 5,000 to 40,000Daltons. In some instances, an amount of the molecular crowding agent isat least about 5% by volume based on the total volume of the hybridizingcomposition. In some instances, an amount of the molecular crowdingagent is less than 50% by volume based on the total volume of thehybridizing composition. In some instances, methods described hereinfurther comprise an additive for controlling a melting temperature ofthe target nucleic acid. In some instances, an amount of the additivefor controlling melting temperature of the target nucleic acid is atleast about 2% by volume based on the total volume of the hybridizingcomposition. In some instances, an amount of the additive forcontrolling melting temperature of the nucleic acid is in the range ofabout 2% to 50% by volume based on the total volume of the hybridizingcomposition. In some instances, the pH buffer comprises at least onebuffering agent selected from the group consisting of Tris, HEPES, TAPS,Tricine, Bicine, Bis-Tris, sodium hydroxide (NaOH), potassium hydroxide(KOH), TES, EPPS, and MOPS. In some instances, the pH buffer furthercomprises a second organic solvent. In some instances, the pH buffercomprises MOPS and methanol. In some instances, an amount of the pHbuffer is effective to maintain the pH of the hybridizing composition tobe in the range of about 3 to about 10. In some instances, the surfacebound nucleic acid is coupled to the surface through covalent ornoncovalent bonding. In some instances, the polymer surface comprisesone or more hydrophilic polymer layers, and the surface bound nucleicacid is coupled to the one or more hydrophilic polymer layers. In someinstances, no more than 10% of the target nucleic acid is associatedwith the surface without hybridizing to the polymer surface boundnucleic acid. In some instances, the one or more hydrophilic polymerlayers comprises a molecule selected from the group consisting ofpolyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA),polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methylmethacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, anddextran. In some instances, the one or more hydrophilic polymer layerscomprises at least one dendrimer.

In some instances, bringing the surface bound nucleic acid molecule intocontact with the hybridizing composition is performed for a period of nomore than 25 minutes. In some instances, bringing the surface boundnucleic acid molecule into contact with the hybridizing composition isperformed for a period of no more than 15 minutes. In some instances,bringing the surface bound nucleic acid molecule into contact with thehybridizing composition is performed for a period of between 2-25minutes. In some instances, the isothermal conditions are at atemperature in the range of about 30 to 70 degrees Celsius. In someinstances, attaching the target nucleic acid molecule to the surfacecomprises hybridizing the target nucleic acid molecule to the surfacebound nucleic acid molecule with a hybridization stringency of at least80%. In some instances, attaching the target nucleic acid molecule tothe surface comprises hybridizing the target nucleic acid molecule tothe surface bound nucleic acid molecule with an increased hybridizationefficiency, as compared to a comparable hybridization reaction whereinthe organic buffer is a saline-sodium citrate and hybridizing isperformed for 120 minutes at 90 degrees Celsius for 5 minutes followedby cooling for 120 minutes to reach a final temperature of 37 degreesCelsius. In some instances, the target nucleic acid molecule is presentin the hybridizing composition at a 1 nanomolar concentration or less.In some instances, the target nucleic acid is present in the hybridizingcomposition at a 250 picomolar concentration or less. In some instances,the target nucleic acid molecule is present in the hybridizingcomposition at a 100 picomolar concentration or less. In some instances,the target nucleic acid molecule is present in the hybridizingcomposition at a 50 picomolar concentration or less. In some instances,methods further comprise hybridizing at least a portion of the surfacebound nucleic acid molecule to at least a portion of the target nucleicacid molecule in the hybridizing composition, wherein hybridizing doesnot consist of cooling. In some instances, bringing the surface boundnucleic acid into contact with the hybridizing composition comprisingthe target nucleic acid is performed under conditions of stringency thatprevent the target nucleic acid molecule from hybridizing to anon-complementary nucleic acid molecule. In some instances, thestringency is at least or about 70%, 80%, or 90%. In some instances, thestringency is at least 80%. Provided herein are methods of attaching atarget nucleic acid molecule to a surface, the method comprising: (a)providing at least one surface bound nucleic acid molecule, wherein theat least one surface bound nucleic acid molecule is coupled to asurface; and (b) bringing a hybridizing composition comprising a targetnucleic acid molecule into contact with the at least one surface boundnucleic acid molecule, wherein the hybridizing composition comprises:(i) at least one organic solvent; and (ii) a pH buffer. In someinstances, the surface exhibits a level of non-specific Cy3 dyeabsorption of less than about 0.25 molecules/μm². In some instances, nomore than 5% of a total number of the target nucleic acid molecule isassociated with the surface without hybridizing to the surface boundnucleic acid molecule. In some instances, bringing the hybridizingcomposition into contact with the at least one surface bound nucleicacid molecule is performed under isothermal conditions. In someinstances, the surface bound nucleic acid molecule is coupled to thesurface by being tethered to the surface. In some instances, the surfaceis a hydrophilic polymer surface. In some instances, the surface has awater contact angle of less than 45 degrees.

In some instances, the at least one organic solvent has a dielectricconstant of no greater than about 115 when measured at 68 degreesFahrenheit. In some instances, the organic solvent is a polar aproticsolvent. In some instances, the organic solvent is an organic solventhaving a dielectric constant of no greater than 40 when measured at 70degrees Fahrenheit. In some instances, the organic solvent isacetonitrile, alcohol, or formamide. In some instances, the organicsolvent comprises at least one functionality selected from hydroxy,nitrile, lactone, sulfone, sulfite, and carbonate. In some instances,the organic solvent is miscible with water. In some instances, theorganic solvent is present in an amount effective to denature a doublestranded nucleic acid. In some instances, an amount of the organicsolvent is at least about 5% by volume based on the total volume of thehybridizing composition. In some instances, an amount of the organicsolvent is in the range of about 5% to 95% by volume based on the totalvolume of the hybridizing composition. In some instances, an amount ofthe pH buffer is no greater than 90% by volume based on the total volumeof the hybridizing composition. In some instances, the hybridizingcomposition further comprises a molecular crowding agent. In someinstances, the molecular crowding agent is selected from the groupconsisting of polyethylene glycol (PEG), dextran, hydroxypropyl methylcellulose (HPMC), hydroxyethyl methyl cellulose (HEMC), hydroxybutylmethyl cellulose, hydroxypropyl cellulose, methycellulose, and hydroxylmethyl cellulose, and any combination thereof. In some instances, themolecular crowding agent is polyethylene glycol (PEG). In someinstances, the molecular crowding agent has a molecular weight in therange of about 5,000 to 40,000 Daltons. In some instances, an amount ofthe molecular crowding agent is at least about 5% by volume based on thetotal volume of the hybridizing composition. In some instances, anamount of the molecular crowding agent is less than 50% by volume basedon the total volume of the hybridizing composition. In some instances,methods further comprise an additive for controlling a meltingtemperature of the target nucleic acid. In some instances, an amount ofthe additive for controlling the melting temperature of the targetnucleic acid molecule is at least about 2% by volume based on the totalvolume of the hybridizing composition. In some instances, an amount ofthe additive for controlling the melting temperature of the nucleic acidis in the range of about 2% to 50% by volume based on the total volumeof the hybridizing composition. In some instances, the pH buffercomprises at least one buffering agent selected from the groupconsisting of Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, sodiumhydroxide (NaOH), potassium hydroxide (KOH), TES, EPPS, and MOPS. Insome instances, the pH buffer further comprises a second organicsolvent. In some instances, the pH buffer comprises MOPS and methanol.In some instances, an amount of the pH buffer is effective to maintainthe pH of the hybridizing composition to be in the range of about 3 toabout 10.

In some instances, the surface bound nucleic acid molecule is coupled tothe surface through covalent or noncovalent bonding. In some instances,the polymer surface comprises one or more hydrophilic polymer layers,wherein the surface bound nucleic acid is coupled to the one or morehydrophilic polymer layers. In some instances, no more than 10% of thetotal number of the target nucleic acid molecule is associated with thesurface without hybridizing to the polymer surface bound nucleic acidmolecule. In some instances, the one or more hydrophilic polymer layerscomprises a molecule selected from the group consisting of polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine),poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA),poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA),poly-lysine, poly-glucoside, streptavidin, and dextran. In someinstances, the one or more hydrophilic polymer layers comprises at leastone dendrimer. In some instances, bringing the surface bound nucleicacid molecule into contact with the hybridizing composition is performedfor a period of no more than 25 minutes. In some instances, bringing thesurface bound nucleic acid molecule into contact with the hybridizingcomposition is performed for a period of no more than 15 minutes. Insome instances, bringing the surface bound nucleic acid molecule intocontact with the hybridizing composition is performed for a periodbetween 2-25 minutes. In some instances, the isothermal conditions areat a temperature in the range of about 30 to 70 degrees Celsius. In someinstances, attaching the target nucleic acid molecule to the surfacecomprises hybridizing the target nucleic acid molecule to the surfacebound nucleic molecule with a hybridization stringency of at least 80%.In some instances, attaching the target nucleic acid molecule to thesurface comprises hybridizing the target nucleic acid molecule to thesurface bound nucleic acid molecule with an increased hybridizationefficiency, as compared to a comparable hybridization reaction whereinthe organic buffer is a saline-sodium citrate and hybridizing isperformed for 120 minutes at 90 degrees Celsius for 5 minutes followedby cooling for 120 minutes to reach a final temperature of 37 degreesCelsius. In some instances, the target nucleic acid molecule is presentin the hybridizing composition at a 1 nanomolar concentration or less.In some instances, the target nucleic acid molecule is present in thehybridizing composition at a 250 picomolar concentration or less. Insome instances, the target nucleic acid molecule is present in thehybridizing composition at a 100 picomolar concentration or less. Insome instances, the target nucleic acid molecule is present in thehybridizing composition at a 50 picomolar concentration or less. In someinstances, methods further comprise hybridizing at least a portion ofthe surface bound nucleic acid molecule to at least a portion of thetarget nucleic acid molecule in the hybridizing composition, whereinhybridizing does not consist of cooling.

Provided herein are methods of sequencing a target nucleic acidmolecule, the methods comprising: (a) bringing a surface bound nucleicacid molecule coupled to a surface into contact with a hybridizingcomposition comprising a target nucleic acid molecule, wherein thehybridizing composition comprises: (i) at least one organic solvent; and(ii) a pH buffer; (b) amplifying the target nucleic acid molecule toform a plurality of clonally-amplified clusters of the target nucleicacid; and (c) determining the identity of the target nucleic acidmolecule, wherein a fluorescence image of the surface comprising theplurality of clonally-amplified clusters of the target nucleic acidmolecule exhibits a contrast-to-noise ratio (CNR) of at least 20 whenthe fluorescence image is captured using a fluorescence imaging systemunder non-signal saturating conditions. In some instances, methodsdescribed herein further comprise hybridizing the target nucleic acidmolecule to the at least one surface bound nucleic acid coupled to thesurface. In some instances, the CNR is at least 50. In some instances,the organic solvent is a polar aprotic solvent. In some instances, theorganic solvent is an organic solvent having a dielectric constant of nogreater than 40 as measured at 70 degrees Fahrenheit. In some instances,the organic solvent is acetonitrile, alcohol, or formamide. In someinstances, the organic solvent comprises at least one functionalityselected from hydroxy, nitrile, lactone, sulfone, sulfite, andcarbonate. In some instances, the organic solvent is miscible withwater. In some instances, the organic solvent is present in an amounteffective to denature a double stranded nucleic acid. In some instances,an amount of the organic solvent is at least about 5% by volume based onthe total volume of the hybridizing composition. In some instances, anamount of the organic solvent is in the range of about 5% to 95% byvolume based on the total volume of the hybridizing composition. In someinstances, an amount of the pH buffer is no greater than 90% by volumebased on the total volume of the hybridizing composition. In someinstances, the hybridizing composition further comprises a molecularcrowding agent. In some instances, the molecular crowding agent isselected from the group consisting of polyethylene glycol (PEG),dextran, hydroxypropyl methyl cellulose (HPMC), hydroxyethyl methylcellulose (HEMC), hydroxybutyl methyl cellulose, hydroxypropylcellulose, methycellulose, and hydroxyl methyl cellulose, and anycombination thereof. In some instances, the molecular crowding agent ispolyethylene glycol (PEG). In some instances, the molecular crowdingagent has a molecular weight in the range of about 5,000 to 40,000Daltons. In some instances, an amount of the molecular crowding agent isat least about 5% by volume based on the total volume of the hybridizingcomposition. In some instances, an amount of the molecular crowdingagent is less than 50% by volume based on the total volume of thehybridizing composition. In some instances, methods described hereinfurther comprise an additive for controlling a melting temperature ofthe target nucleic acid molecule. In some instances, an amount of theadditive for controlling melting temperature of the target nucleic acidis at least about 2% by volume based on the total volume of thehybridizing composition. In some instances, an amount of the additivefor controlling melting temperature of the nucleic acid molecule is inthe range of about 2% to 50% by volume based on the total volume of thehybridizing composition. In some instances, the pH buffer comprises atleast one buffering agent selected from the group consisting of Tris,HEPES, TAPS, Tricine, Bicine, Bis-Tris, sodium hydroxide (NaOH),potassium hydroxide (KOH), TES, EPPS, and MOPS. In some instances, thepH buffer further comprises a second organic solvent. In some instances,the pH buffer comprises MOPS and methanol. In some instances, an amountof the pH buffer is effective to maintain the pH of the hybridizingcomposition to be in the range of about 3 to about 10.

In some instances, the surface bound nucleic acid molecule is coupled tothe surface through covalent or noncovalent bonding. In some instances,the polymer surface comprises one or more hydrophilic polymer layers,wherein the surface bound nucleic acid molecule is coupled to the one ormore hydrophilic polymer layers. In some instances, the polymer surfaceexhibits a level of non-specific Cyanine3 (Cy3) dye absorption of lessthan about 0.25 molecules per micrometer squared (μm²). In someinstances, no more than 5% of a total number of the target nucleic acidmolecule is associated with the surface without hybridizing to thesurface bound nucleic acid molecule. In some instances, no more than 10%of the total number of the target nucleic acid molecule is associatedwith the surface without hybridizing to the surface bound nucleic acidmolecule. In some instances, the one or more hydrophilic polymer layerscomprises a molecule selected from the group consisting of polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine),poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA),poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA),poly-lysine, poly-glucoside, streptavidin, and dextran. In someinstances, the one or more hydrophilic polymer layers comprise at leastone dendrimer. In some instances, bringing the surface bound nucleicacid molecule into contact with the hybridizing composition is performedunder isothermal conditions. In some instances, bringing the surfacebound nucleic acid molecule into contact with the hybridizingcomposition is performed at a temperature in the range of about 30 to 70degrees Celsius. In some instances, bringing the surface bound nucleicacid molecule into contact with the hybridizing composition is performedfor a period of no more than 25 minutes. In some instances, methodsdescribed herein further comprise removing the hybridizing compositionfrom the surface after the period of no more than 25 minutes. In someinstances, bringing the surface bound nucleic acid molecule into contactwith the hybridizing composition is performed for a period between 2-25minutes. In some instances, bringing the surface bound nucleic acidmolecule into contact with the hybridizing composition is performed fora period between 2-4 minutes. In some instances, bringing the surfacebound nucleic acid molecule into contact with the hybridizingcomposition is performed for a period of 2 minutes. In some instances,the at least one surface bound nucleic acid molecule is circular. Insome instances, methods further comprise hybridizing at least a portionof the surface bound nucleic acid molecule to at least a portion of thetarget nucleic acid in the hybridizing composition, which hybridizingdoes not consist of cooling. In some instances, bringing the surfacebound nucleic acid into contact with the hybridizing compositioncomprising the target nucleic acid is performed under conditions ofstringency that prevent the target nucleic acid from hybridizing to anon-complementary nucleic acid. In some instances, the stringency is atleast or about 70%, 80%, or 90%. In some instances, the stringency is atleast 80%.

Provided herein are compositions for hybridizing a target nucleic acidmolecule to a surface bound nucleic acid molecule, the compositionscomprising: (a) a target nucleic acid molecule; (b) at least one organicsolvent; and (c) a pH buffer. In some instances, no more than 10% of atotal number of the target nucleic acid molecule is associated with thesurface without hybridizing to the surface bound nucleic acid molecule.In some instances, no more than 5% of the total number of the targetnucleic acid molecule is bound to the surface without hybridizing to thesurface bound nucleic acid molecule.

In some instances, the organic solvent is a polar aprotic solvent. Insome instances, the organic solvent is an organic solvent having adielectric constant of no greater than 40 when measured at 70 degreesFahrenheit. In some instances, the organic solvent is acetonitrile,alcohol, or formamide. In some instances, the organic solvent comprisesat least one functionality selected from hydroxy, nitrile, lactone,sulfone, sulfite, and carbonate. In some instances, the organic solventis miscible with water. In some instances, the organic solvent ispresent in an amount effective to denature a double stranded nucleicacid. In some instances, an amount of the organic solvent is at leastabout 5% by volume based on the total volume of the composition. In someinstances, an amount of the organic solvent is in the range of about 5%to 95% by volume based on the total volume of the composition. In someinstances, the pH buffer system comprises a pH buffer. In someinstances, an amount of the pH buffer is no greater than 90% by volumebased on the total volume of the composition. In some instances, thecomposition further comprises a molecular crowding agent. In someinstances, the molecular crowding agent is selected from the groupconsisting of polyethylene glycol (PEG), dextran, hydroxypropyl methylcellulose (HPMC), hydroxyethyl methyl cellulose (HEMC), hydroxybutylmethyl cellulose, hydroxypropyl cellulose, methycellulose, and hydroxylmethyl cellulose, and any combination thereof. In some instances, themolecular crowding agent is polyethylene glycol (PEG). In someinstances, the molecular crowding agent has a molecular weight in therange of about 5,000 to 40,000 Daltons. In some instances, an amount ofthe molecular crowding agent is at least about 5% by volume based on thetotal volume of the composition. In some instances, an amount of themolecular crowding agent is less than 50% by volume based on the totalvolume of the composition. In some instances, the compositions forhybridizing a target nucleic acid molecule to a surface bound nucleicacid molecule further comprise an additive for controlling a meltingtemperature of the target nucleic acid molecule. In some instances, anamount of the additive for controlling the melting temperature of thetarget nucleic acid molecule is at least about 2% by volume based on thetotal volume of the composition. In some instances, an amount of theadditive for controlling the melting temperature of the nucleic acidmolecule is in the range of about 2% to 50% by volume based on the totalvolume of the composition. In some instances, the pH buffer comprises atleast one buffering agent selected from the group consisting of Tris,HEPES, TAPS, Tricine, Bicine, Bis-Tris, sodium hydroxide (NaOH),potassium hydroxide (KOH), TES, EPPS, and MOPS. In some instances, thepH buffer further comprises a second organic solvent. In some instances,the pH buffer comprises MOPS and methanol. In some instances, an amountof the pH buffer is effective to maintain the pH of the composition tobe in the range of about 3 to about 10.

In some instances, the surface bound nucleic acid molecule is coupled toa surface through covalent or noncovalent bonding. In some instances,the surface is a hydrophilic polymer surface. In some instances, thepolymer surface comprises one or more hydrophilic polymer layers,wherein the surface bound nucleic acid molecule is coupled to the one ormore hydrophilic polymer layers. In some instances, the one or morehydrophilic polymer layers comprises a molecule selected from the groupconsisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA),poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid)(PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methylmethacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, anddextran. In some instances, the one or more hydrophilic polymer layerscomprises at least one dendrimer. In some instances, the target nucleicacid molecule is present in the composition at a 1 nanomolarconcentration or less. In some instances, the target nucleic acidmolecule is present in the composition at a 250 picomolar concentrationor less. In some instances, the target nucleic acid molecule is presentin the composition at a 100 picomolar concentration or less. In someinstances, the target nucleic acid molecule is present in thecomposition at a 50 picomolar concentration or less.

Provided herein, in some instances, are microfluidic systems, comprisingthe compositions described herein. In some instances, the microfluidicsystems comprise a flow cell device. In some instances, the flow celldevice is a microfluidic chip flow cell. In some instances, the flowcell device is a capillary flow cell device. In some instances, at leastone surface of the flow cell device comprises one or more hydrophilicpolymer layers comprising a molecule selected from the group consistingof polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA),polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methylmethacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, anddextran. In some instances, the flow cell device comprises a compositiondescribed herein formulated as a fluid. In some instances, the flow celldevice comprises one or more surface bound nucleic acid moleculescoupled to the at least one surface of the flow cell. In some instances,a target nucleic acid molecule in the composition is hybridized to theone or more surface bound nucleic acid molecules coupled to the at leastone surface of the flow cell. In some instances, the flow cell device isoperatively coupled to an imaging system configured to capture an imageof the at least one surface of the flow cell comprising the hybridizedtarget nucleic acid molecule and the one or more surface bound nucleicacid molecules. Methods described herein comprise determining anidentity of the target nucleic acid molecule using the microfluidicsystems described herein.

Provided herein are kits comprising: (a) a surface; and (b) acomposition comprising: (i) at least one organic solvent; and (ii) a pHbuffer. In some instances, the surface comprises one or more surfacebound nucleic acid molecules coupled to the surface. In some instances,the surface is a hydrophilic polymer surface. In some instances, thesurface has a water contact angle of less than 45 degrees. In someinstances, the hydrophilic polymer surface comprises one or morehydrophilic polymer layers, and wherein the surface bound nucleic acidis coupled to the one or more hydrophilic polymer layers. In someinstances, the one or more hydrophilic polymer layers comprises amolecule selected from the group consisting of polyethylene glycol(PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA),poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA),poly-lysine, poly-glucoside, streptavidin, and dextran. In someinstances, the kit further comprises instructions for hybridizing theone or more surface bound nucleic acid molecules to one or more targetnucleic acid molecules. In some instances, the kit further comprisesinstructions for determining the identity of the one or more targetnucleic acid molecules.

In some instances, the organic solvent is a polar aprotic solvent. Insome instances, the organic solvent is an organic solvent having adielectric constant of no greater than 40 when measured at 70 degreesFahrenheit. In some instances, the organic solvent is acetonitrile,alcohol, or formamide. In some instances, the organic solvent comprisesat least one functionality selected from hydroxy, nitrile, lactone,sulfone, sulfite, and carbonate. In some instances, the organic solventis miscible with water. In some instances, the organic solvent ispresent in an amount effective to denature a double stranded nucleicacid. In some instances, an amount of the organic solvent is at leastabout 5% by volume based on the total volume of the composition. In someinstances, an amount of the organic solvent is in the range of about 5%to 95% by volume based on the total volume of the composition. In someinstances, the pH buffer system comprises a pH buffer. In someinstances, an amount of the pH buffer is no greater than 90% by volumebased on the total volume of the composition. In some instances, thecomposition further comprises a molecular crowding agent. In someinstances, the molecular crowding agent is selected from the groupconsisting of polyethylene glycol (PEG), dextran, hydroxypropyl methylcellulose (HPMC), hydroxyethyl methyl cellulose (HEMC), hydroxybutylmethyl cellulose, hydroxypropyl cellulose, methycellulose, and hydroxylmethyl cellulose, and any combination thereof. In some instances, themolecular crowding agent is polyethylene glycol (PEG). In someinstances, the molecular crowding agent has a molecular weight in therange of about 5,000 to 40,000 Daltons. In some instances, an amount ofthe molecular crowding agent is at least about 5% by volume based on thetotal volume of the composition. In some instances, an amount of themolecular crowding agent is less than 50% by volume based on the totalvolume of the composition. In some instances, the compositions forhybridizing a target nucleic acid molecule to a surface bound nucleicacid molecule further comprise an additive for controlling a meltingtemperature of the one or more target nucleic acid molecules. In someinstances, an amount of the additive for controlling melting temperatureof the one or more target nucleic molecules acid is at least about 2% byvolume based on the total volume of the composition. In some instances,an amount of the additive for controlling melting temperature of thenucleic acid is in the range of about 2% to 50% by volume based on thetotal volume of the composition. In some instances, the pH buffercomprises at least one buffering agent selected from the groupconsisting of Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, sodiumhydroxide (NaOH), potassium hydroxide (KOH), TES, EPPS, and MOPS. Insome instances, the pH buffer further comprises a second organicsolvent. In some instances, the pH buffer comprises MOPS and methanol.In some instances, an amount of the pH buffer is effective to maintainthe pH of the composition to be in the range of about 3 to about 10.

Provided herein are methods of using the kits described herein. In someinstances, the surface bound nucleic acid molecules is coupled to thesurface by a covalent or a noncovalent bond. In some instances, themethods comprise: (a) combining the one or more target nucleic acidmolecules and the composition of the kit to form a master mix; and (b)bringing the master mix into contact with the one or more surface boundnucleic acid molecules coupled to the surface provided in the kit. Insome instances, the methods further comprise (c) hybridizing the one ormore target nucleic acid molecules with the one or more surface boundnucleic acid molecules coupled to the surface. In some instances, thesurface exhibits a level of non-specific Cy3 dye absorption of less thanabout 0.25 molecules/μm². In some instances, no more than 10% of a totalnumber of the one or more target nucleic acid molecules is associatedwith the surface without hybridizing to the surface bound nucleic acidmolecule. In some instances, no more than 5% of the total number of theone or more target nucleic acid molecules is associated with the surfacewithout hybridizing to the one or more surface bound nucleic acidmolecules. In some instances, hybridizing the one or more target nucleicacid molecules with the one or more surface bound nucleic acid moleculescoupled to the surface is performed under isothermal conditions. In someinstances, the isothermal conditions are performed at a temperature in arange of 30 to 70 degrees Celsius. In some instances, the methodsfurther comprise (d) amplifying the target nucleic acid hybridized tothe surface bound nucleic acid to form a plurality of clonally-amplifiedclusters of the one or more target nucleic acid molecules coupled to thesurface; and (c) determining the identity of the one or more targetnucleic acid molecules. In some instances, a fluorescence image of thesurface comprising the plurality of clonally-amplified clusters of theone or more target nucleic acid molecules exhibits a contrast-to-noiseratio (CNR) of at least 20 when the fluorescence image is captured usinga fluorescence imaging system under non-signal saturating conditions. Insome instances, the CNR is at least 50.

In some instances, hybridizing the surface bound nucleic acid and thetarget nucleic acid is performed for a period of no more than 25minutes. In some instances, methods of using the kits described hereinfurther comprise removing the composition from the surface after theperiod of no more than 25 minutes. In some instances, hybridizing thesurface bound nucleic acid and the target nucleic acid is performed fora period between 2-25 minutes. In some instances, hybridizing the one ormore surface bound nucleic acid molecules and the one or more targetnucleic acid molecules is performed for a period of between 2-4 minutes.In some instances, hybridizing the one or more surface bound nucleicacid molecules and the one or more target nucleic acid molecules isperformed for a period of 2 minutes. In some instances, the at least onesurface bound nucleic acid is circular. In some instances, hybridizingdoes not consist of cooling. In some instances, bringing the master mixinto contact with the one or more surface bound nucleic acid moleculesis performed under conditions of stringency that prevent the one or moretarget nucleic acid molecules from hybridizing to a non-complementarynucleic acid. In some instances, the stringency is at least or about70%, 80%, or 90%. In some instances, the stringency is at least 80%.

Provided herein are systems comprising: (a) a surface comprising one ormore surface bound nucleic acids molecules coupled to the surface; (b)one or more target nucleic acid molecules; and (c) a compositioncomprising: (i) at least one organic solvent; and (ii) a pH buffer. Insome instances, the systems further comprise a fluorescence imagingdevice. In some instances, the surface is a hydrophilic polymer surface.In some instances, the surface has a water contact angle of less than 45degrees. In some instances, the hydrophilic polymer surface comprisesone or more hydrophilic polymer layers, wherein the one or more surfacebound nucleic acid molecules is coupled to the one or more hydrophilicpolymer layers. In some instances, the one or more hydrophilic polymerlayers comprises a molecule selected from the group consisting ofpolyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA),polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methylmethacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA),poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA),polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, anddextran.

In some instances, the organic solvent is an organic solvent having adielectric constant of no greater than 40 when measured at 70 degreesFahrenheit. In some instances, the organic solvent is acetonitrile,alcohol, or formamide. In some instances, the organic solvent comprisesat least one functionality selected from hydroxy, nitrile, lactone,sulfone, sulfite, and carbonate. In some instances, the organic solventis miscible with water. In some instances, the organic solvent ispresent in an amount effective to denature a double stranded nucleicacid. In some instances, an amount of the organic solvent is at leastabout 5% by volume based on the total volume of the composition. In someinstances, an amount of the organic solvent is in the range of about 5%to 95% by volume based on the total volume of the composition. In someinstances, the pH buffer system comprises a pH buffer. In someinstances, an amount of the pH buffer is no greater than 90% by volumebased on the total volume of the composition. In some instances, thecomposition further comprises a molecular crowding agent. In someinstances, the molecular crowding agent is selected from the groupconsisting of polyethylene glycol (PEG), dextran, hydroxypropyl methylcellulose (HPMC), hydroxyethyl methyl cellulose (HEMC), hydroxybutylmethyl cellulose, hydroxypropyl cellulose, methyl cellulose, andhydroxyl methyl cellulose, and any combination thereof. In someinstances, the molecular crowding agent is polyethylene glycol (PEG). Insome instances, the molecular crowding agent has a molecular weight inthe range of about 5,000 to 40,000 Daltons. In some instances, an amountof the molecular crowding agent is at least about 5% by volume based onthe total volume of the composition. In some instances, an amount of themolecular crowding agent is less than 50% by volume based on the totalvolume of the composition. In some instances, the systems describedherein further comprise an additive for controlling a meltingtemperature of the target nucleic acid. In some instances, an amount ofthe additive for controlling melting temperature of the one or moretarget nucleic acid molecules is at least about 2% by volume based onthe total volume of the composition. In some instances, an amount of theadditive for controlling melting temperature of the one or more nucleicacid molecules is in the range of about 2% to 50% by volume based on thetotal volume of the composition. In some instances, the pH buffercomprises at least one buffering agent selected from the groupconsisting of Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, sodiumhydroxide (NaOH), potassium hydroxide (KOH), TES, EPPS, and MOPS. Insome instances, the pH buffer further comprises a second organicsolvent. In some instances, the pH buffer comprises MOPS and methanol.In some instances, an amount of the pH buffer is effective to maintainthe pH of the composition to be in the range of about 3 to about 10.

Provided herein are methods of using the systems described herein. Insome instances, the one or more surface bound nucleic acid molecules iscoupled to the surface by a covalent or a noncovalent bond. In someinstances, the methods comprise: (a) combining the one or more targetnucleic acid molecules and the composition of the system to form amaster mix; (b) bringing the master mix into contact with the one ormore surface bound nucleic acid molecules coupled to the surfaceprovided in the system; (c) hybridizing the one or more target nucleicacid molecules with the one or more surface bound nucleic acid moleculescoupled to the surface; (d) amplifying the one or more target nucleicacid molecules hybridized to the one or more surface bound nucleic acidmolecules to form a plurality of clonally-amplified clusters of the oneor more target nucleic acid molecules coupled to the surface; and (e)determining the identity of the one or more target nucleic acidmolecules by capturing an image of the surface with the fluorescenceimaging device. In some instances, the surface exhibits a level ofnon-specific Cy3 dye absorption of less than about 0.25 molecules/μm².In some instances, hybridizing the one or more target nucleic acidmolecules with the one or more surface bound nucleic acid moleculescoupled to the surface is performed under isothermal conditions. In someinstances, the isothermal conditions are performed at a temperature in arange of 30 to 70 degrees Celsius. In some instances, no more than 10%of a total number of the one or more target nucleic acid molecules isassociated with the surface without hybridizing to the one or moresurface bound nucleic acid molecules. In some instances, no more than 5%of the total number of the one or more target nucleic acid molecules isassociated with the surface without hybridizing to the one or moresurface bound nucleic acid molecules. In some instances, a fluorescenceimage of the surface comprising the amplified one or more target nucleicacid molecules exhibits a contrast-to-noise ratio (CNR) of at least 20when the fluorescence image is captured using the fluorescence imagingdevice under non-signal saturating conditions. In some instances, theCNR is at least 50.

In some instances, hybridizing the one or more surface bound nucleicacid molecules and the one or more target nucleic acid molecules isperformed for a period of no more than 25 minutes. In some instances,the methods disclosed herein further comprise removing the compositionfrom the surface after the period of no more than 25 minutes. In someinstances, hybridizing the one or more surface bound nucleic acidmolecules and the one or more target nucleic acid molecules is performedfor a period between 2-25 minutes. In some instances, hybridizing theone or more surface bound nucleic acid molecules and the one or moretarget nucleic acid molecules is performed for a period between 2-4minutes. In some instances, hybridizing the one or more surface boundnucleic acid molecules and the one or more target nucleic acid moleculesis performed for a period of 2 minutes. In some instances, the at leastone surface bound nucleic acid is circular. In some instances,hybridizing does not consist of cooling. In some instances, bringing theone or more surface bound nucleic acid molecules into contact with thehybridizing composition comprising the one or more target nucleic acidmolecules is performed under conditions of stringency that prevent theone or more target nucleic acid molecules from hybridizing to anon-complementary nucleic acid molecule. In some instances, thestringency is at least or about 70%, 80%, or 90%. In some instances, thestringency is at least 80%.

Illustrative Embodiment 3

Disclosed herein are methods of determining an identity of a nucleotidein a target nucleic acid sequence comprising: (a) providing acomposition comprising: (i) two or more copies of said target nucleicacid sequence; (ii) two or more primer nucleic acid molecules that arecomplementary to one or more regions of said target nucleic acidsequence; and (iii) two or more polymerase molecules; (b) contactingsaid composition with a polymer nucleotide conjugate under conditionssufficient to allow a multivalent binding complex to be formed betweensaid polymer-nucleotide conjugate and said two or more copies of saidtarget nucleic acid sequence in said composition of (a), wherein thepolymer-nucleotide conjugate comprises two or more copies of anucleotide moiety and optionally one or more detectable labels; and (c)detecting said multivalent binding complex, thereby determining theidentity of said nucleotide in the target nucleic acid sequence. In someinstances, the target nucleic acid sequence is DNA. In some instances,the detection of the multivalent binding complex is performed in theabsence of unbound or solution-borne polymer nucleotide conjugates. Insome instances, the target nucleic acid sequence has been replicated oramplified or has been produced by replication or amplification. In someinstances, the one or more detectable labels are fluorescent labels. Insome instances, detecting the multivalent complex comprises afluorescence measurement. In some instances, the contacting comprisesuse of one type of polymer-nucleotide conjugate. In some instances, thecontacting comprises use of two or more types of polymer-nucleotideconjugates. In some instances, each type of the two or more types ofpolymer-nucleotide conjugate comprises a different type of nucleotidemoiety. In some instances, the contacting comprises use of three typesof polymer-nucleotide conjugates, wherein each type of the three typesof polymer-nucleotide conjugates comprises a different type ofnucleotide moiety. In some instances, the polymer-nucleotide conjugatecomprises a blocked nucleotide moiety. In some instances, the blockednucleotide is a 3′-O-azidomethyl nucleotide, a 3′-O-methyl nucleotide,or a 3′-O-alkyl hydroxylamine nucleotide. In some instances, saidcontacting occurs in the presence of an ion that stabilizes saidmultivalent binding complex. In some instances, the contacting is donein the presence of strontium ions, magnesium ions, calcium ions, or anycombination thereof. In some instances, the polymerase molecules arecatalytically inactive. In some instances, the polymerase molecules havebeen rendered catalytically inactive by mutation or chemicalmodification. In some instances, the polymerase molecules have beenrendered catalytically inactive by the absence of a necessary ion orcofactor. In some instances, the polymerase molecules are catalyticallyactive. In some instances, the polymer-nucleotide conjugate does notcomprise a blocked nucleotide moiety. In some instances, the multivalentbinding complex has a persistence time of greater than 2 seconds. Insome instances, the method can be carried out at a temperature within arange of 25° C. to 62° C. In some instances, the polymer-nucleotideconjugate further comprises one or more fluorescent labels and the twoor more copies of the target nucleic acid sequence are deposited on,attached to, or hybridized to a surface, wherein a fluorescence image ofthe multivalent binding complex on the surface has a contrast to noiseratio in the detecting step of greater than 20. In some instances, thecomposition of (a) is deposited on a surface using a buffer thatincorporates a polar aprotic solvent. In some instances, the contactingis performed under a condition that stabilizes said multivalent bindingcomplex when said nucleotide moiety is complementary to a next base ofsaid target nucleic acid sequence and destabilizes said multivalentbinding complex when said nucleotide moiety is not complementary to saidnext base of said target nucleic acid sequence. In some instances, saidpolymer-nucleotide conjugate comprises a polymer having a plurality ofbranches and said two or more nucleotide moieties are attached to saidbranches. In some instances, said polymer has a star, comb,cross-linked, bottle brush, or dendrimer configuration. In someinstances, said polymer-nucleotide conjugate comprises one or morebinding groups selected from the group consisting of an avidin, abiotin, an affinity tag, and combinations thereof. In some instances,the method further comprises a dissociation step that destabilizes saidmultivalent binding complex formed between the composition of (a) andthe polymer-nucleotide conjugate, said dissociation step enablingremoval of said polymer-nucleotide conjugate. In some instances, themethod further comprises an extension step to incorporate a nucleotidethat is complementary to a next base of the target nucleic acid sequenceinto said two or more primer nucleic acid molecules. In some instances,the extension step occurs concurrently with or after said dissociationstep.

Disclosed herein are methods of determining an identity of a nucleotidein a target nucleic acid sequence comprising: (a) providing acomposition comprising: (i) two or more copies of said target nucleicacid sequence; (ii) two or more primer nucleic acid molecules that arecomplementary to one or more regions of said target nucleic acidsequence; and (ii). two or more polymerase molecules; (b) contactingsaid composition with a polymer nucleotide conjugate under conditionssufficient to allow a multivalent complex to be formed between saidpolymer-nucleotide conjugate and said two or more copies of said targetnucleic acid sequence in said composition of (a), wherein thepolymer-nucleotide conjugate comprises two or more copies of areversibly terminated nucleotide moiety and optionally one or morecleavable detectable labels; and (c) detecting said multivalent complex,thereby determining the identity of said nucleotide in the targetnucleic acid sequence. In some instances, the target nucleic acidsequence is DNA. In some instances, the method further comprisescontacting the composition of (a) with reversibly terminated nucleotidesor polymer-nucleotide conjugates comprising two or more copies of areversibly terminated nucleotide following the detection of saidmultivalent binding complex. In some instances, the target nucleic acidsequence has been replicated or amplified or has been produced byreplication or amplification. In some instances, the one or moredetectable labels are fluorescent labels. In some instances, detectingthe multivalent complex comprises a fluorescence measurement. In someinstances, the contacting comprises use of one type ofpolymer-nucleotide conjugate. In some instances, the contactingcomprises use of two or more types of polymer-nucleotide conjugates. Insome instances, each type of the two or more types of polymer-nucleotideconjugate comprises a different type of nucleotide moiety. In someinstances, the contacting comprises use of three types ofpolymer-nucleotide conjugate, wherein each type of the three types ofpolymer-nucleotide conjugate comprises a different type of nucleotidemoiety. In some instances, the polymer-nucleotide conjugate comprises ablocked nucleotide moiety. In some instances, the blocked nucleotide isa 3′-O-azidomethyl, 3′-O-methyl, or 3′-O-alkyl hydroxylamine. In someinstances, said contacting occurs in the presence of an ion thatstabilizes said multivalent binding complex. In some instances, thepolymerase molecules are catalytically inactive. In some instances, thepolymerase molecules have been rendered catalytically inactive bymutation or chemical modification. In some instances, the polymerasemolecules are catalytically active. In some instances, thepolymer-nucleotide conjugate does not comprise a blocked nucleotidemoiety. In some instances, the method can be carried out at atemperature within a range of 25° C. to 80° C. In some instances, thepolymer-nucleotide conjugate further comprises one or more fluorescentlabels and the two or more copies of the target nucleic acid sequenceare deposited on, attached to, or hybridized to a surface, wherein afluorescence image of the multivalent binding complex on the surface hasa contrast to noise ratio in the detecting step of greater than 20.

Also disclosed herein are systems comprising: (a) one or more computerprocessors individually or collectively programmed to implement a methodcomprising: (i) contacting a substrate comprising multiple copies of atarget nucleic acid sequence tethered to a surface of the substrate witha reagent comprising a polymerase and one or more primer nucleic acidsequences that are complementary to one or more regions of said targetnucleic acid sequence to form a primed target nucleic acid sequence;(ii) contacting the substrate surface with a reagent comprising apolymer nucleotide conjugate under conditions sufficient to allow amultivalent binding complex to be formed between said polymer-nucleotideconjugate and two or more copies of said primed target nucleic acidsequence, wherein the polymer-nucleotide conjugate comprises two or morecopies of a known nucleotide moiety and a detectable label; (iii)acquiring and processing an image of the substrate surface to detectsaid multivalent binding complex, thereby determining the identity of anucleotide in the target nucleic acid sequence. In some instances, thesystem further comprises a fluidics module configured to deliver aseries of reagents to the substrate surface in a specified sequence andfor specified time intervals. In some instances, the system furthercomprises an imaging module configured to acquire images of thesubstrate surface. In some instances, (ii) and (iii) are repeated two ormore times thereby determining the identity of a series of two or morenucleotides in the target nucleic acid sequence. In some instances, theseries of steps further comprise a dissociation step that destabilizessaid multivalent binding complex, said dissociation step enablingremoval of said polymer-nucleotide conjugate. In some instances, theseries of steps further comprises an extension step to incorporate anucleotide that is complementary to a next base of the target nucleicacid sequence into said two or more primer nucleic acid molecules. Insome instances, the extension step occurs concurrently with or aftersaid dissociation step. In some instances, the detectable labelcomprises a fluorophore and the images comprise fluorescence images. Insome instances, the fluorescence images of the multivalent bindingcomplex on the substrate surface has a contrast-to-noise ratio ofgreater than 20 when the fluorophore is cyanine dye 3 (Cy3) and theimage is acquired using an inverted fluorescence microscope equippedwith a 20× objective, NA=0.75, dichroic mirror optimized for 532 nmlight, a bandpass filter optimized for Cyanine dye-3 emission, and acamera, under non-signal saturating conditions while the surface isimmersed in 25 mM ACES, pH 7.4 buffer. In some instances, the series ofsteps is completed in less than 60 minutes. In some instances, theseries of steps is completed in less than 30 minutes. In some instances,the series of steps is completed in less than 10 minutes. In someinstances, an accuracy of base-calling is characterized by a Q-score ofgreater than 25 for at least 80% of the nucleotide identitiesdetermined. In some instances, an accuracy of base-calling ischaracterized by a Q-score of greater than 30 for at least 80% of thenucleotide identities determined. In some instances, an accuracy ofbase-calling is characterized by a Q-score of greater than 40 for atleast 80% of the nucleotide identities determined.

Disclosed herein are compositions comprising: a) a polymer core; and b)two or more nucleotide, nucleotide analog, nucleoside, or nucleosideanalog moieties attached to the polymer core; wherein the length of thelinker is dependent on the nucleotide, nucleotide analog, nucleoside, ornucleoside analog moiety that is attached to the polymer core. Alsodisclosed herein are compositions comprising: a) a mixture ofpolymer-nucleotide conjugates, wherein each polymer-nucleotide conjugatecomprises: i) a polymer core; and ii) two or more nucleotide, nucleotideanalog, nucleoside, or nucleoside analog moieties attached to thepolymer core, wherein the length of the linker is dependent on thenucleotide, nucleotide analog, nucleoside, or nucleoside analog moietythat is attached to the polymer core; and wherein the mixture comprisespolymer-nucleotide conjugates having at least two different types ofattached nucleotides, nucleotide analogs, nucleosides, or nucleosideanalog moieties. In some instances, the polymer core comprises a polymerhaving a plurality of branches and the two or more nucleotide,nucleotide analog, nucleoside, or nucleoside analog moieties areattached to said branches. In some instances, polymer has a star, comb,cross-linked, bottle brush, or dendrimer configuration. In someinstances, the polymer-nucleotide conjugate comprises one or morebinding groups selected from the group consisting of an avidin, abiotin, an affinity tag, and combinations thereof. In some instances,the polymer core comprises a branched polyethylene glycol (PEG)molecule. In some instances, the polymer-nucleotide conjugate comprisesa blocked nucleotide moiety. In some instances, the blocked nucleotideis a 3′-O-azidomethyl nucleotide, a 3′-O-methyl nucleotide, or a3′-O-alkyl hydroxylamine nucleotide. In some instances, thepolymer-nucleotide conjugate further comprises one or more fluorescentlabels.

In some instances, the present disclosure provides methods ofdetermining the identity of a nucleotide in a target nucleic acidcomprising the steps, without regard to any particular order ofoperations, 1) providing a composition comprising: a target nucleic acidcomprising two or more repeats of an identical sequence; two or moreprimer nucleic acids complementary to one or more regions of said targetnucleic acid; and two or more polymerase molecules; 2) contacting saidcomposition with a multivalent binding or incorporation compositioncomprising a polymer-nucleotide conjugate under conditions sufficient toallow a binding or incorporated complex to be formed between saidpolymer-nucleotide conjugate and the composition of step (a), whereinthe polymer-nucleotide conjugate comprises two or more copies of anucleotide and optionally one or more detectable labels; and 3)detecting said binding or incorporated complex, thereby establishing theidentity of said nucleotide in the target nucleic acid polymer. In somefurther instances, the present disclosure provides said method, whereinthe target nucleic acid is DNA, and/or wherein the target nucleic acidhas been replicated, such as by any commonly practiced method of DNAreplication or amplification, such as rolling circle amplification,bridge amplification, helicase dependent amplification, isothermalbridge amplification, rolling circle multiple displacement amplification(RCA/MDA), and/or recombinase based methods of replication oramplification. In some further instances, the present disclosureprovides said method, wherein the detectable label is a fluorescentlabel and/or wherein detecting the complex comprises a fluorescencemeasurement. In some further instances, the present disclosure providessaid method wherein the multivalent binding composition comprises onetype of polymer-nucleotide conjugate, wherein the multivalent bindingcomposition comprises two or more types of polymer-nucleotideconjugates, and/or wherein each type of the two or more types ofpolymer-nucleotide conjugates comprises a different type of nucleotide.In some instances, the present disclosure provides said method whereinthe binding complex or incorporated complex further comprises a blockednucleotide, especially wherein the blocked nucleotide is a3′-O-azidomethyl nucleotide, a 3′-O-alkyl hydroxylamino nucleotide, or a3′-O-methyl nucleotide. In some further instances, the presentdisclosure provides said method wherein the contacting is done in thepresence of strontium ions, barium, magnesium ions, and/or calcium ions.In some instances, the present disclosure provides said method whereinthe polymerase molecule is catalytically inactive, such as where thepolymerase molecule been rendered catalytically inactive by mutation, bychemical modification, or by the absence of a necessary ion or cofactor.In some instances, the present disclosure also provides said methodwherein the polymerase molecule is catalytically active, and/or whereinthe binding complex does not comprise a blocked nucleotide. In someinstances, the present disclosure provides said method wherein thebinding complex has a persistence time of greater than 2 seconds and/orwherein the method is or may be carried out at a temperature of at orabove 15° C., at or above 20° C., at or above 25° C., at or above 35°C., at or above 37° C., at or above 42° C., at or above 55° C., at orabove 60° C., or at or above 72° C., or within a range defined by any ofthe foregoing. In some instances, the present disclosure provides saidmethod wherein the binding complex is deposited on, attached to, orhybridized to, a surface showing a contrast to noise ratio in thedetecting step of greater than 20. In some instances, the presentdisclosure provides said method wherein the composition is depositedunder buffer conditions incorporating a polar aprotic solvent. In someinstances, the present disclosure provides said method wherein thecontacting is performed under a condition that stabilizes said bindingcomplex when said nucleotide is complementary to a next base of saidtarget nucleic acid and destabilizes said binding complex when saidnucleotide is not complementary to said next base of said target nucleicacid. In some instances, the present disclosure provides said methodwherein said polymer-nucleotide conjugate comprises a polymer having aplurality of branches and said plurality of copies of said firstnucleotide are attached to said branches, especially wherein said firstpolymer has a star, comb, cross-linked, bottle brush, or dendrimerconfiguration. In some instances, the present disclosure provides saidmethod wherein said polymer-nucleotide conjugate comprises one or morebinding groups selected from the group consisting of avidin, biotin,affinity tag, and combinations thereof. In some instances, the presentdisclosure provides said method further comprising a dissociation stepthat destabilizes said binding complex formed between the composition of(a) and the polymer-nucleotide conjugate to remove saidpolymer-nucleotide conjugate. In some instances, the present disclosureprovides said method further comprising an extension step to incorporateinto said primer nucleic acid a nucleotide that is complementary to saidnext base of the target nucleic acid, and optionally wherein theextension step occurs currently as or after said dissociation step.

In some instances, the present disclosure provides a compositioncomprising a branched polymer having two or more branches and two ormore copies of a nucleotide, wherein said nucleotide is attached to afirst plurality of said branches or arms, and optionally, wherein one ormore interaction moieties are attached to a second plurality of saidbranches or arms. In some instances, said composition may furthercomprise one or more labels on the polymer. In some instances, thepresent disclosure provides said composition wherein the nucleoside hasa surface density of at least 4 nucleotides per polymer. In someinstances, the present disclosure provides said composition comprisingor incorporating a nucleotide or nucleotide analog that is modified soas to prevent its incorporation into an extending nucleic acid chainduring a polymerase reaction. In some instances, said composition maycomprise or incorporate a nucleotide or nucleotide analog that isreversibly modified so as to prevent its incorporation into an extendingnucleic acid chain during a polymerase reaction. In some instances, thepresent disclosure provides said composition wherein one or more labelscomprise a fluorescent label, a FRET donor, and/or a FRET acceptor. Insome instances, said composition may comprise 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 or more branches or arms, or 2, 4, 8, 16, 32,64, or more, branches or arms. In some instances, the branches or armsmay radiate from a central moiety. In some instances, said compositionmay comprise one or more interaction moieties, which interactionmoieties may comprise avidin or streptavidin; a biotin moiety; anaffinity tag; an enzyme, antibody, minibody, receptor, or other protein;a non-protein tag; a metal affinity tag, or any combination thereof. Insome instances, the present disclosure provides said composition whereinthe polymer comprises polyethylene glycol, polypropylene glycol,polyvinyl acetate, polylactic acid, or polyglycolic acid. In someinstances, the present disclosure provides said composition wherein thenucleotide or nucleotide analog is attached to the branch or arm througha linker; and especially wherein the linker comprises PEG, and whereinthe PEG linker moiety has an average molecular weight of about 1K Da,about 2K Da, about 3K Da, about 4K Da, about 5K Da, about 10K Da, about15K Da, about 20K Da, about 50K Da, about 100K Da, about 150K Da, orabout 200K Da, or greater than about 200K Da. In some instances, thepresent disclosure provides said composition wherein the linkercomprises PEG, and wherein the PEG linker moiety has an averagemolecular weight of between about 5K Da and about 20K Da. In someinstances, the present disclosure provides said composition wherein atleast one nucleotide or nucleotide analog comprises adeoxyribonucleotide, a ribonucleotide, a deoxyribonucleoside, or aribonucleoside; and/or wherein the nucleotide or nucleotide analog isconjugated to the linker through the 5′ end of the nucleotide ornucleotide analog. In some instances, the present disclosure providessaid composition wherein one of the nucleotides or nucleotide analogscomprises deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine,deoxycytidine, adenosine, guanosine, 5-methyl-uridine, and/or cytidine;and wherein the length of the linker is between 1 nm and 1,000 nm. Insome instances, the present disclosure provides said composition whereinat least one nucleotide or nucleotide analog is a nucleotide that hasbeen modified to inhibit elongation during a polymerase reaction or asequencing reaction, such as wherein the at least one nucleotide ornucleotide analog is a nucleotide that lacks a 3′ hydroxyl group; anucleotide that has been modified to contain a blocking group at the 3′position; and/or a nucleotide that has been modified with a 3′-O-azidogroup, a 3′-O-azidomethyl group, a 3′-O-alkyl hydroxylamino group, a3′-phosphorothioate group, a 3′-O-malonyl group, or a 3′-O-benzyl group.In some instances, the present disclosure provides said compositionwherein at least one nucleotide or nucleotide analog is a nucleotidethat has not been modified at the 3′ position.

In some instances, the present disclosure provides a method ofdetermining the sequence of a nucleic acid molecule comprising thesteps, without regard to any particular order, of 1) providing a nucleicacid molecule comprising a template strand and a complementary strandthat is at least partially complementary to the template strand; 2)contacting the nucleic acid molecule with the one or more nucleic acidbinding composition according to any of the examples disclosed herein;3) detecting binding of the nucleic acid binding composition to thenucleic acid molecule, and 4) determining an identity of a terminalnucleotide to be incorporated into said complementary strand of saidnucleic acid molecule. In some instances, the present disclosureprovides a method of determining the sequence of a nucleic acid moleculecomprising the steps, without regard to any particular order, of 1)providing a nucleic acid molecule comprising a template strand and acomplementary strand that is at least partially complementary to thetemplate strand; 2) contacting the nucleic acid molecule with the one ormore nucleic acid binding compositions according to any of the examplesdisclosed herein; 3) detecting partial or complete incorporation of thenucleic acid binding composition to the nucleic acid molecule, and 4)determining an identity of a terminal nucleotide to be incorporated intosaid complementary strand of said nucleic acid molecule from the partialor complete incorporation of the examples described herein. In someinstances, the present disclosure provides said method furthercomprising incorporating said terminal nucleotide into saidcomplementary strand, and repeating said contacting, detecting, andincorporating steps for one or more additional iterations, therebydetermining the sequence of said template strand of said nucleic acidmolecule. In some instances, the present disclosure provides saidmethod, wherein said nucleic acid molecule is tethered to a solidsupport; and, in some examples, wherein the solid support comprises aglass or polymer substrate, at least one hydrophilic polymer coatinglayer, and a plurality of oligonucleotide molecules attached to at leastone hydrophilic polymer coating layer. In some instances, the presentdisclosure provides said method, further comprising examples wherein atleast one hydrophilic polymer coating layer comprises PEG; and/orwherein at least one hydrophilic polymer layer comprises a branchedhydrophilic polymer having at least 8 branches. In some instances, thepresent disclosure provides said method, wherein the plurality ofoligonucleotide molecules is present at a surface density of at least500 molecules/mm², at least 1,000 molecules/mm², at least 5,000molecules/mm², at least 10,000 molecules/mm², at least 20,000molecules/mm², at least 50,000 molecules/mm², at least 100,000molecules/mm², or at least 500,000 molecules/mm². In some instances, thepresent disclosure provides said method, wherein said nucleic acidmolecule has been clonally-amplified on a solid support. In someinstances, the present disclosure provides said method, wherein theclonal amplification comprises the use of a polymerase chain reaction(PCR), multiple displacement amplification (MDA), transcription-mediatedamplification (TMA), nucleic acid sequence-based amplification (NASBA),strand displacement amplification (SDA), real-time SDA, bridgeamplification, isothermal bridge amplification, rolling circleamplification (RCA), circle-to-circle amplification, helicase-dependentamplification, recombinase-dependent amplification, single-strandedbinding (SSB) protein-dependent amplification, or any combinationthereof. In some instances, the present disclosure provides said method,wherein the one or more nucleic acid binding compositions are labeledwith fluorophores and the detecting step comprises use of fluorescenceimaging; and especially wherein the fluorescence imaging comprises dualwavelength excitation/four wavelength emission fluorescence imaging. Insome instances, the present disclosure provides said method, whereinfour different nucleic acid binding compositions, each comprising adifferent nucleotide or nucleotide analog, are used to determine theidentity of the terminal nucleotide, wherein the four different nucleicacid binding compositions are labeled with separate respectivefluorophores, and wherein the detecting step comprises simultaneousexcitation at a wavelength sufficient to excite all four fluorophoresand imaging of fluorescence emission at wavelengths sufficient to detecteach respective fluorophore. In some instances, the present disclosureprovides said method, wherein four different nucleic acid bindingcompositions, each comprising a different nucleotide or nucleotideanalog, are used to determine the identity of the terminal nucleotide,wherein the four different nucleic acid binding compositions are labeledwith cyanine dye 3 (Cy3), cyanine dye 3.5 (Cy3.5), cyanine dye 5 (Cy5),and cyanine dye 5.5. (Cy5.5) respectively, and wherein the detectingstep comprises simultaneous excitation at any two of 532 nm, 568 nm and633 nm, and imaging of fluorescence emission at about 570 nm, 592 nm,670 nm, and 702 nm respectively; and/or wherein the fluorescence imagingcomprises dual wavelength excitation/dual wavelength emissionfluorescence imaging. In some instances, the present disclosure providessaid method, wherein four different nucleic acid binding compositions,each comprising a different nucleotide or nucleotide analog, are used todetermine the identity of the terminal nucleotide, wherein one, two,three, or four different nucleic acid binding compositions arerespectively labeled, each with a with distinct fluorophore or set offluorophores, and wherein the detecting step comprises simultaneousexcitation at a wavelength sufficient to excite one, two, three, or fourfluorophores or sets of fluorophores, and imaging of fluorescenceemission at wavelengths sufficient to detect each respectivefluorophore. In some instances, the present disclosure provides saidmethod, wherein three different nucleic acid binding or incorporationcompositions, each comprising a different nucleotide or nucleotideanalog, are used to determine the identity of the terminal nucleotide,wherein one, two, or three different nucleic acid binding orincorporation compositions are respectively labeled, each with a withdistinct fluorophore or set of fluorophores, and wherein the detectingstep comprises simultaneous excitation at a wavelength sufficient toexcite one, two, or three, fluorophores or sets of fluorophores, andimaging of fluorescence emission at wavelengths sufficient to detecteach respective fluorophore, and wherein detection of the fourthnucleotide is determined or determinable with reference to the locationof “dark” or unlabeled spots or target nucleotides. In some instances,the present disclosure provides said method, wherein the multivalentbinding or incorporation composition may comprise three types ofpolymer-nucleotide conjugates and wherein each type of the three typesof polymer-nucleotide conjugates comprises a different type ofnucleotide. In some instances, the present disclosure provides saidmethod, wherein the detection of the binding or incorporation complex isperformed in the absence of unbound or solution-borne polymer nucleotideconjugates.

In some instances, the present disclosure provides said method, whereinfour different nucleic acid binding compositions, or three differentnucleic acid binding or incorporation compositions, each comprising adifferent nucleotide or nucleotide analog, are used to determine theidentity of the terminal nucleotide, wherein one of the four or threedifferent nucleic acid binding or incorporation compositions is labeledwith a first fluorophore, one is labeled with a second fluorophore, oneis labeled with both the first and second fluorophore, and one is notlabeled or is absent, and wherein the detecting step comprisessimultaneous excitation at a first excitation wavelength and a secondexcitation wavelength and images are acquired at a first fluorescenceemission wavelength and a second fluorescence emission wavelength. Insome instances, the present disclosure provides said method, wherein thefirst fluorophore is Cy3, the second fluorophore is Cy5, the firstexcitation wavelength is 532 nm or 568 nm, the second excitationwavelength is 633 nm, the first fluorescence emission wavelength isabout 570 nm, and the second fluorescence emission wavelength is about670 nm. In some instances, the present disclosure provides said method,wherein the detection label can comprise one or more portions of afluorescence resonance energy transfer (FRET) pair, such that multipleclassifications can be performed under a single excitation and imagingstep. In some instances, the present disclosure provides said method,wherein a sequencing reaction cycle comprising the contacting,detecting, and incorporating/extending steps is performed in less than30 minutes in less than 20 minutes, or in less than 10 minutes. In someinstances, the present disclosure provides said method, wherein anaverage Q-score for base calling accuracy over a sequencing run isgreater than or equal to 30, and/or greater than or equal to 40. In someinstances, the present disclosure provides said method, wherein at least50%, at least 60%, at least 70%, at least 80%, or at least 90% of theterminal nucleotides identified have a Q-score of greater than 30 and/orgreater than or equal to 40. In some instances, the present disclosureprovides said method, herein at least 95% of the terminal nucleotidesidentified have a Q-score of greater than 30.

In some instances, the present disclosure provides a reagent comprisingone or more nucleic acid binding compositions as disclosed herein and abuffer. For example, in some instances, the present disclosure providesa reagent, wherein said reagent comprises 1, 2, 3, 4, or more nucleicacid binding or incorporation compositions, wherein each nucleic acidbinding or incorporation composition comprises a single type ofnucleotide. In some instances, a reagent of the present disclosurecomprises 1, 2, 3, 4, or more nucleic acid binding or incorporationcompositions, wherein each nucleic acid binding or incorporationcomposition comprises a single type of nucleotide or nucleotide analog,and wherein said nucleotide or nucleotide analog may respectivelycorrespond to one or more from the group consisting of adenosinetriphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate(AMP), deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate(dADP), and deoxyadenosine monophosphate (dAMP); one or more from thegroup consisting of thymidine triphosphate (TTP), thymidine diphosphate(TDP), thymidine monophosphate (TMP), deoxythymidine triphosphate(dTTP), deoxythymidine diphosphate (dTDP), deoxythymidine monophosphate(dTMP), uridine triphosphate (UTP), uridine diphosphate (UDP), uridinemonophosphate (UMP), deoxyuridine triphosphate (dUTP), deoxyuridinediphosphate (dUDP), and deoxyuridine monophosphate (dUMP); one or morefrom the group consisting of cytidine triphosphate (CTP), cytidinediphosphate (CDP), cytidine monophosphate (CMP), deoxycytidinetriphosphate (dCTP), deoxycytidine diphosphate (dCDP), and deoxycytidinemonophosphate (dCMP); and one or more from the group consisting ofguanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosinemonophosphate (GMP), deoxyguanosine triphosphate (dGTP), deoxyguanosinediphosphate (dGDP), and deoxyguanosine monophosphate (dGMP). In someother examples or some further examples, the present disclosure providesa reagent comprising or further comprising 1, 2, 3, 4, or more nucleicacid binding or incorporation compositions, wherein each nucleic acidbinding or incorporation composition comprises a single type ofnucleotide or nucleotide analog, and wherein said nucleotide ornucleotide analog may respectively correspond to one or more from thegroup consisting of ATP, ADP, AMP, dATP, dADP, dAMP, TTP, TDP, TMP,dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP,dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, and dGMP.

Disclosed herein are kits comprising any of the nucleic acid binding orincorporation compositions disclosed herein and/or any of the reagentsdisclosed herein, and/or one or more buffers; and instructions for theuse thereof.

Disclosed herein are systems for performing any of the methods disclosedherein, comprising any of the nucleic acid binding or incorporationcompositions disclosed herein, and/or any of the reagents disclosedherein. In some instances, a system is configured to iteratively performthe sequential contacting of tethered, primed nucleic acid moleculeswith said nucleic acid binding or incorporation compositions and/or saidreagents; and for the detection of binding or incorporation of thedisclosed nucleic acid binding or incorporation compositions to the oneor more primed nucleic acid molecules.

In some instances, the present disclosure provides a compositioncomprising a particle (e.g., a nanoparticle or polymer core), saidparticle comprising a plurality of enzyme or protein binding orincorporation substrates, wherein the enzyme or protein binding orincorporation substrates bind with one or more enzymes or proteins toform one or more binding or incorporation complexes (e.g., a multivalentbinding or incorporation complex), and wherein said binding orincorporation may be monitored or identified by observation of thelocation, presence, or persistence of the one or more binding orincorporation complexes. In some instances, said particle may comprise apolymer, branched polymer, dendrimer, liposome, micelle, nanoparticle,or quantum dot. In some instances, said substrate may comprise anucleotide, a nucleoside, a nucleotide analog, or a nucleoside analog.In some instances, the enzyme or protein binding or incorporationsubstrate may comprise an agent that can bind with a polymerase. In someinstances, the enzyme or protein may comprise a polymerase. In someinstances, said observation of the location, presence, or persistence ofone or more binding or incorporation complexes may comprise fluorescencedetection. In some instances, the present disclosure provides acomposition comprising multiple distinct particles as disclosed herein,wherein each particle comprises a single type of nucleoside ornucleoside analog, and wherein each nucleoside or nucleoside analog isassociated with a fluorescent label of a detectably different emissionor excitation wavelength. In some instances, the present disclosureprovides said composition further comprising one or more labels, e.g.,fluorescence labels, on the particle. In some instances, the presentdisclosure provides said composition wherein the composition comprisesat least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or more than 20 tetherednucleotides, nucleotide analogs, nucleosides, or nucleoside analogstethered to the particle. In some instances, the present disclosureprovides said composition wherein the nucleoside or nucleoside analog ispresent at a surface density of between 0.001 and 1,000,000 per μm²,between 0.01 and 1,000,000 per μm², between 0.1 and 1,000,000 per μm²,between 1 and 1,000,000 per μm², between 10 and 1,000,000 per μm²,between 100 and 1,000,000 per μm², between 1,000 and 1,000,000 per μm²,between 1,000 and 100,000 per μm², between 10,000 and 100,000 per μm²,or between 50,000 and 100,000 per μm², or within a range defined by anytwo of the foregoing values. In some instances, the present disclosureprovides said composition wherein the nucleoside or nucleoside analog ispresent within a nucleotide or nucleotide analog. In some instances, thepresent disclosure provides said composition wherein the compositioncomprises or incorporates a nucleotide or nucleotide analog that ismodified so as to prevent its incorporation into an extending nucleicacid chain during a polymerase reaction. In some instances, the presentdisclosure provides said composition wherein the composition comprisesor incorporates a nucleotide or nucleotide analog that is reversiblymodified so as to prevent its incorporation into an extending nucleicacid chain during a polymerase reaction. In some instances, the presentdisclosure provides said composition wherein one or more labels comprisea fluorescent label, a FRET donor, and/or a FRET acceptor. In someinstances, the present disclosure provides said composition wherein thesubstrate (e.g., nucleotide, nucleotide analog, nucleoside, ornucleoside analog) is attached to the particle through a linker. In someinstances, the present disclosure provides said composition wherein atleast one nucleotide or nucleotide analog is a nucleotide that has beenmodified to inhibit elongation during a polymerase reaction or asequencing reaction, such as, for example, a nucleotide that lacks a 3′hydroxyl group; a nucleotide that has been modified to contain ablocking group at the 3′ position; a nucleotide that has been modifiedwith a 3′-O-azido group, a 3′-O-azidomethyl group, a 3′-O-alkylhydroxylamino group, a 3′-phosphorothioate group, a 3′-O-malonyl group,or a 3′-O-benzyl group; and/or a nucleotide that has not been modifiedat the 3′ position.

In some instances, the present disclosure provides a method ofdetermining the sequence of a nucleic acid molecule comprising thesteps, without regard to order, of 1) providing a nucleic acid moleculecomprising a template strand and a complementary strand that is at leastpartially complementary to the template strand; 2) contacting thenucleic acid molecule with the one or more nucleic acid binding orincorporation composition according to any of the instances disclosedherein; 3) detecting binding or incorporation of the nucleic acidbinding or incorporation composition to the nucleic acid molecule, and4) determining an identity of a terminal nucleotide to be incorporatedinto said complementary strand of said nucleic acid molecule. In someinstances, said method may further comprise incorporating said terminalnucleotide into said complementary strand, and repeating saidcontacting, detecting, and incorporating steps for one or moreadditional iterations, thereby determining the sequence of said templatestrand of said nucleic acid molecule. In some instances, the presentdisclosure provides said method wherein said nucleic acid molecule hasbeen clonally-amplified on a solid support. In some instances, thepresent disclosure provides said method wherein the clonal amplificationcomprises the use of a polymerase chain reaction (PCR), multipledisplacement amplification (MDA), transcription-mediated amplification(TMA), nucleic acid sequence-based amplification (NASBA), stranddisplacement amplification (SDA), real-time SDA, bridge amplification,isothermal bridge amplification, rolling circle amplification,circle-to-circle amplification, helicase-dependent amplification,recombinase-dependent amplification, single-stranded binding (SSB)protein-dependent amplification, or any combination thereof. In someinstances, the present disclosure provides said method wherein asequencing reaction cycle comprising the contacting, detecting, andincorporating steps is performed in less than 30 minutes, less than 20minutes, or in less than 10 minutes. In some instances, the presentdisclosure provides said method wherein an average Q-score for basecalling accuracy over a sequencing run is greater than or equal to 30,or greater than or equal to 40. In some instances, the presentdisclosure provides said method wherein at least 50%, at least 60%, atleast 70%, at least 80%, or at least 90% of the terminal nucleotidesidentified have a Q-score of greater than 30; or greater than 40. Insome instances, the present disclosure provides said method wherein atleast 95% of the terminal nucleotides identified have a Q-score ofgreater than 30.

In some instances, the present disclosure provides a reagent comprisingone or more nucleic acid binding or incorporation compositions asdisclosed herein, and a buffer. In some instances, the presentdisclosure provides said reagent, wherein said reagent comprises 1, 2,3, 4, or more nucleic acid binding or incorporation compositions,wherein each nucleic acid binding or incorporation composition comprisesa single type of nucleotide or nucleotide analog, and wherein saidnucleotide or nucleotide analog comprises a nucleotide, nucleotideanalog, nucleoside, or nucleoside analog. In some instances, the presentdisclosure provides said method wherein said reagent comprises 1, 2, 3,4, or more nucleic acid binding or incorporation compositions, whereineach nucleic acid binding or incorporation composition comprises asingle type of nucleotide or nucleotide analog, and wherein saidnucleotide or nucleotide analog may respectively correspond to one ormore from the group consisting of ATP, ADP, AMP, dATP, dADP, and dAMP;one or more from the group consisting of TTP, TDP, TMP, dTTP, dTDP,dTMP, UTP, UDP, UMP, dUTP, dUDP, and dUMP; one or more from the groupconsisting of CTP, CDP, CMP, dCTP, dCDP, and dCMP; and one or more fromthe group consisting of GTP, GDP, GMP, dGTP, dGDP, and dGMP. In someinstances, the present disclosure provides said method wherein saidreagent comprises 1, 2, 3, 4, or more nucleic acid binding orincorporation compositions, wherein each nucleic acid binding orincorporation composition comprises a single type of nucleotide ornucleotide analog, and wherein said nucleotide or nucleotide analog mayrespectively correspond to one or more from the group consisting of ATP,ADP, AMP, dATP, dADP, dAMP TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP,UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP,dGTP, dGDP, and dGMP.

In some instances, the present disclosure provides a kit comprising anyof the compositions disclosed herein; and/or any of the reagentsdisclosed herein; one or more buffers; and instructions for the usethereof.

In some instances, the present disclosure provides a system forperforming any of the methods disclosed herein; wherein said methods maycomprise use of any of the compositions as disclosed herein; and/or anyof the reagents as disclosed herein; one or more buffers, and one ormore nucleic acid molecules optionally tethered or attached to a solidsupport, wherein said system is configured to iteratively perform forthe sequential contacting of said nucleic acid molecules with saidcomposition and/or said reagent; and for the detection of binding orincorporation of the nucleic acid binding or incorporation compositionsto the one or more nucleic acid molecules.

In some instances, the present disclosure provides a composition asdisclosed herein for use in increasing the contrast to noise ratio (CNR)of a labeled nucleic acid complex bound to or associated with a surface.

In some instances, the present disclosure provides a composition asdisclosed herein for use in establishing or maintaining control over thepersistence time of a signal from a labeled nucleic acid complex boundto or associated with a surface.

In some instances, the present disclosure provides a composition asdisclosed herein for use in establishing or maintaining control over thepersistence time of a fluorescence, luminescence, electrical,electrochemical, colorimetric, radioactive, magnetic, or electromagneticsignal from a labeled nucleic acid complex bound to or associated with asurface.

In some instances, the present disclosure provides a composition asdisclosed herein for use in increasing the specificity, accuracy, orread length of a nucleic acid sequencing and/or genotyping application.

In some instances, the present disclosure provides a composition asdisclosed herein for use in increasing the specificity, accuracy, orread length in a sequencing by binding or incorporation, sequencing bysynthesis, single molecule sequencing, or ensemble sequencing method.

In some instances, the present disclosure provides a reagent asdisclosed herein for use in increasing the contrast to noise ratio (CNR)of a labeled nucleic acid complex bound to or associated with a surface.

In some instances, the present disclosure provides a reagent asdisclosed herein for use in establishing or maintaining control over thepersistence time of a signal from a labeled nucleic acid complex boundto or associated with a surface.

In some instances, the present disclosure provides a reagent asdisclosed herein for use in establishing or maintaining control over thepersistence time of a fluorescence, luminescence, electrical,electrochemical, colorimetric, radioactive, magnetic, or electromagneticsignal from a labeled nucleic acid complex bound to or associated with asurface.

In some instances, the present disclosure provides a reagent asdisclosed herein for use in increasing the specificity, accuracy, orread length of a nucleic acid sequencing and/or genotyping application.

In some instances, the present disclosure provides a reagent asdisclosed herein for use in increasing the specificity, accuracy, orread length in a sequencing by binding or incorporation, sequencing bysynthesis, single molecule sequencing, or ensemble sequencing method.

Definitions

Unless otherwise defined, all of the technical terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart in the field to which this disclosure belongs.

As used in this specification and the appended enumerated embodiments,the singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise. Any reference to “or” herein isintended to encompass “and/or” unless otherwise stated.

As used herein, the term “about” a number refers to that number plus orminus 10% of that number. The term “about” when used in the context of arange refers to that range minus 10% of its lowest value and plus 10% ofits greatest value.

As used herein, the terms “DNA hybridization” and “nucleic acidhybridization” are used interchangeably and are intended to cover anytype of nucleic acid hybridization, e.g., DNA hybridization or RNAhybridization, unless otherwise specified.

As used herein, the term “isothermal” refers to a condition in which thetemperature remains substantially constant. A temperature that is“substantially constant” may deviate (e.g., increase or decrease) over aperiod of time by no more than 0.25 degrees, 0.50 degrees, 0.75 degrees,or 1.0 degrees.

The terms “anneal” or “hybridize,” are used herein interchangeably torefer to the ability of two nucleic acid molecules to combine together.In some cases, the “combining” refers to Watson-Crick base pairingbetween the bases in each of the two nucleic acid molecules.

As used herein, “hybridization specificity” refers to a measure of theability of nucleic acid molecules (e.g., adapter sequences, primersequences, or oligonucleotide sequences) to correctly hybridize to aregion of a target nucleic acid molecule with a nucleic acid sequencethat is completely complementary to the nucleic acid molecule.

As used herein, “hybridization sensitivity” refers to a concentrationrange of sample (or target) nucleic molecules in which hybridizationoccurs with high specificity. In some cases, as little as 50 picomolarconcentration of sample nucleic acid molecules in which hybridizationwith high specify is achieved with the methods, compositions, systemsand kits described herein. In some instances, the range is between about1 nanomolar to about 50 picomolar concentrations of sample nucleic acidmolecules.

As used herein, “hybridization efficiency” refers to a measure of thepercentage of total available nucleic acid molecules (e.g., adaptersequences, primer sequences, or oligonucleotide sequences) that arehybridized to the region of the target nucleic acid molecule with thenucleic acid sequence that is completely complementary to the nucleicacid molecule.

As used herein, the term “hybridization stringency” refer to apercentage of nucleotide bases within at least a portion of a nucleicacid sequence undergoing a hybridization (e.g., a hybridization region)reaction that is complementary through standard Watson-Crick basepairing. In a non-limiting example, a hybridization stringency of 80%means that a stable duplex can be formed in which 80% of thehybridization region undergoes Watson-Crick base pairing. A higherhybridization stringency means a higher degree of Watson-Crick basepairing is required in a given hybridization reaction in order to form astable duplex.

As used herein, the terms, “isolate” and “purify,” are usedinterchangeably herein unless specified otherwise.

As used herein, “nucleic acid” (also referred to as a “polynucleotide”,“oligonucleotide”, ribonucleic acid (RNA), or deoxyribonucleic acid(DNA)) is a linear polymer of two or more nucleotides joined by covalentinternucleosidic linkages, or variants or functional fragments thereof.In naturally occurring examples of nucleic acids, the internucleosidelinkage is a phosphodiester bond. However, other examples optionallycomprise other internucleoside linkages, such as phosphorothiolatelinkages and may or may not comprise a phosphate group. Nucleic acidsinclude double- and single-stranded DNA, as well as double- andsingle-stranded RNA, DNA/RNA hybrids, peptide-nucleic acids (PNAs),hybrids between PNAs and DNA or RNA, and may also include other types ofnucleic acid modifications.

As used herein, a “nucleotide” refers to a nucleotide, nucleoside, oranalog thereof. The nucleotide refers to both naturally occurring andchemically modified nucleotides and can include but are not limited to anucleoside, a ribonucleotide, a deoxyribonucleotide, a protein-nucleicacid residue, or derivatives. Examples of the nucleotide includes anadenine, a thymine, a uracil, a cytosine, a guanine, or residue thereof;a deoxyadenine, a deoxythymine, a deoxyuracil, a deoxycytosine, adeoxyguanine, or residue thereof; a adenine PNA, a thymine PNA, a uracilPNA, a cytosine PNA, a guanine PNA, or residue or equivalents thereof,an N- or C-glycoside of a purine or pyrimidine base (e.g., adeoxyribonucleoside containing 2-deoxy-D-ribose or ribonucleosidecontaining D-ribose).

“Complementary,” as used herein, refers to the topological compatibilityor matching together of interacting surfaces of a ligand molecule andits receptor. Thus, the receptor and its ligand can be described ascomplementary, and furthermore, the contact surface characteristics arecomplementary to each other.

“Branched polymer”, as used herein, refers to a polymer having aplurality of functional groups that help conjugate a biologically activemolecule such as a nucleotide, and the functional group can be either onthe side chain of the polymer or directly attached to a central core orcentral backbone of the polymer. The branched polymer can have a linearbackbone with one or more functional groups coming off the backbone forconjugation. The branched polymer can also be a polymer having one ormore sidechains, wherein the one or more side chains has a site suitablefor conjugation. Examples of the functional group include but arelimited to hydroxyl, ester, amine, carbonate, acetal, aldehyde, aldehydehydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone,hydrazide, thiol, alkanoic acid, acid halide, isocyanate,isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, andtresylate.

“Polymerase,” as used herein, refers to an enzyme that contains anucleotide binding moiety and helps formation of a binding complexbetween a target nucleic acid and a complementary nucleotide. Thepolymerase can have one or more activities including, but not limitedto, base analog detection activities, DNA polymerization activity,reverse transcriptase activity, DNA binding or incorporation, stranddisplacement activity, and nucleotide binding or incorporation andrecognition. The polymerase can include catalytically inactivepolymerase, catalytically active polymerase, reverse transcriptase, andother enzymes containing a nucleotide binding or incorporation moiety.

“Persistence time,” as used herein, refers to the length of time that abinding complex, which is formed between the target nucleic acid, apolymerase, and a conjugated or unconjugated nucleotide, remains stablewithout any binding component dissociating from the binding complex. Thepersistence time is indicative of the stability of the binding complexand strength of the binding interactions. Persistence time can bemeasured by observing the onset and/or duration of a binding complex,such as by observing a signal from a labeled component of the bindingcomplex. For example, a labeled nucleotide or a labeled reagentcomprising one or more nucleotides may be present in a binding complex,thus allowing the signal from the label to be detected during thepersistence time of the binding complex. One non-limiting example oflabel is a fluorescent label.

Abbreviations

Dimethyl sulfoxide (DMSO),

Dimethyl formamide (DMF),

3-(N-morpholino)propanesulfonic acid (MOPS),

Acetonitrile (ACN)

2-(N-morpholino)ethanesulfonic acid (MES)

saline-sodium citrate (SSC)

Formamide (Form.)

Tris(hydroxymethyl)aminomethane (Tris)

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the examples and enumerated embodiments providedherein.

Example 1—DNA Hybridization on Low Non-Specific Binding Surface

FIGS. 1A-B provide examples of the optimized hybridization achieved onlow binding surface using the disclosed hybridization method (FIG. 1A)with reduced concentrations of hybridization reporter probe andshortened hybridization times, as compared to the results achieved usinga traditional hybridization protocol on the same low binding surface(FIG. 1B).

FIG. 1A shows hybridization reactions on the low binding surfaceaccording to the embodiments described herein. The rows provide two testhybridization conditions, hybridization condition 1 (“Hyb 1”) andhybridization condition 2 (“Hyb 2”). Hyb 1 refers to the hybridizationbuffer composition C10 from Table 1. Hyb 2 refers to the hybridizationbuffer composition D18 from Table 1. A hybridization reporter probe(complementary oligonucleotide sequences labeled with a Cy™3 fluorophoreat the 5′ end) at concentrations reported in FIG. 1A (10 nM, 1 nM, 250pM, 100 pM, and 50 pM) were hybridized in the buffer compositions at 60degrees Celsius for 2 minutes.

FIG. 1B shows hybridization reactions on the low binding surfaceaccording to a standard hybridization protocol with standardhybridization conditions (“Standard Hyb Conditions”). A standardhybridization buffer of 2×-5× saline-sodium citrate (SSC) was used withsame hybridization reporter probe above at the same concentrationsabove, as shown in FIG. 1A. The standard hybridization reaction wasperformed at 90 degrees Celsius with a slow cool process (2 hours) toreach 37 degrees Celsius.

For each hybridization reaction provided in FIG. 1A and FIG. 1B, the toprow for each hybridization reaction is test (“T”), which is thecomplementary oligos (e.g., CY3™-5′-ACCCTGAAAGTACGTGCATTACATG-3′ (SEQ IDNO: 3)), and the bottom row for each hybridization reach is a control(“C”), which is a noncomplementary oligos (e.g.,CY3™-5′-ATGTCTATTACGTCACACTATTATG-3′ (SEQ ID NO: 4)).

The surfaces used for all testing conditions were ultra-low non-specificbinding surfaces having a level of non-specific Cy3 dye absorptioncorresponding to less than or equal to about 0.25 molecules/μm². In thisexample, the low non-specific binding surfaces used were a glasssubstrates that were functionalized with Silane-PEG-5K-COOH (NanocsInc.).

Following completion of the hybridization reactions, wells were washedwith 50 mM Tris pH 8.0; 50 mM NaCl.

Images were obtained using an inverted microscope (Olympus IX83)equipped with 100×TIRF objective, NA=1.4 (Olympus), dichroic mirroroptimized for 532 nm light (Semrock, Di03-R532-t1-25×36), a bandpassfilter optimized for Cy3 emission, (Semrock, FF01-562/40-25), and acamera (sCMOS, Andor Zyla) under non-signal saturating conditions for 1s, (Laser Quantum, Gem 532, <1 W/cm² at the sample) while the sample wasimmersed in a buffer (25 mM ACES, pH 7.4 buffer). Images were collectedas described above, and the results are shown in FIG. 1A (optimized) andFIG. 1B (standard).

A significant signal was observed from the reaction with 250 picomolar(pM) in both Hyb 1 and Hyb 2 hybridization reactions (FIG. 1A), ascompared with the negative control. In contrast, no signal was observedfrom the reaction with 250 pM in the Standard Hyb conditions, ascompared with the negative control. The same result was observed forlower input concentrations (e.g., 100 pM, 50 pM) of the hybridizationreporter probe. FIG. 1A shows more than 200-fold decrease in input DNA(labeled oligo) required for specific DNA capture on low non-specificbinding surfaces tested, a 50× decrease in hybridization times, and areduction in the hybridization temperatures by half, as compared withstandard hybridization methods and reagents on the same low non-specificbinding substrates (FIG. 1B). The buffer compositions and methodsdescribed herein boast improved hybridization specificity, decreasedworkflow times and increased hybridization sensitivity.

Example 2

Buffer compositions according to various embodiments described hereinwere optimized to facilitate hybridization of monotemplateoligonucleotide fragments to the low non-specific binding surfacedescribed herein.

Preparing the low non-specific binding surfaces. Glass substrates (175um 22×60 mm², Corning Glass) were cleaned with KOH and ethanol. Lowbinding glass surfaces were prepared by incubating Silane-PEG5K-NHS(Nanocs) in ethanol at 65 degrees for 30 minutes. Oligonucleotides with5′ modified NH₂ were grafted to these surfaces in a mixture of 1micromolar (uM), 5.1 μM, and 46 uM oligonucleotides inmethanol/phosphate buffer for 20 minutes, to form immobilizedoligonucleotides coupled to the glass substrates.

Circularizing monotemplate oligonucleotide fragments into library.Monotemplate oligonucleotide fragments (approximately 100 base pairs inlength) were circularized using splint ligation protocol that containedcomplementary fragments to surface grafted primers.

Hybridizing the circularized library to immobilized oligonucleotides.Following circularization of the library, circular library fragmentswere added at a concentration of 100 picomolar (pM) in various testhybridization test mixtures indicated by rows B-F. Individualbuffer/library hybridization mixtures were added to 384 well plate withthe functionalized surface affixed at 50 degrees Celsius for 4 minutes.

Visualizing hybridization using test buffer compositions. IntercalatingDNA stain was added to the buffer/library hybridization mixturesfollowing the hybridization reaction to visualize the hybridization ofthe circularized libraries. The 384 well plate was imaged using afluorescence microscope and 488 nanometer (nm) excitation with a 60×water immersion objective (1.2 NA, Olympus) (See FIG. 3). A number ofbuffer compositions were tested for the hybridization of target nucleicacid (e.g., circularized library) with surface bound nucleic acid (e.g.,immobilized oligonucleotides). Table 1 provides the buffer compositionsand immobilized oligonucleotide concentrations for each reaction seen inFIG. 3, with columns 10-21 in Table 1 corresponding with columns 10-21of FIG. 3, and rows B-F corresponding to row B-F of FIG. 3. F10 and F11are negative controls using standard hybridization conditions, where nobackground signal was detected signifying both the validity of thenegative control and the low non-specific binding nature of surfacestested.

TABLE 1 Buffer compositions tested for hybridizing target nucleic acidwith surface bound nucleic acid Graft concentration 1 uM 9 10 11 12 1314 15 B Cracked 75% ACN + 75% ACN + 2xSSC 25% ACN + Std buf. + 30% PEGMES Phos 2xSSC + 10% 5% PEG + PEG 30% Form. C 1 uM 50% ACN + 50% ACN +4xSSC 25% ACN + Std buf. + 20% PEG + 31-NH2-Cy3 MES Tris MES + 20% 10%PEG + 2xSSC PEG + 10% 5% Form. Form. D 1 uM 25% ACN + 25% ACN + 10xSSC50% EtOH + Std buf. + 10% PEG + 31-NH2-Cy3 MES + 2xSSC Tris + 2xSSC2xSSC 10% PEG + 2xSSC + 5% 10% Form. Form. E 1 uM MES + 1xSSC Tris +1xSSC 20xSSC 50% EtOH + Std buf. + 5% Form. + 31-NH2-Cy3 2xSSC + 10% 20%PEG + 2xSSC PEG 10% Form. F 10 nM 10 nM 10 nM 10xSSC + Std Std buf. +10% Form. + 31-NH2-Cy3 31-NH2-Cy3 31-NH2-Cy3 10% Form. 10% Form. 2xSSCGraft concentration 1 uM 5.1 uM 46 uM 16 17 18 19 20 21 B Std 50% ACN +Std Std Std Std 50% Std buf. C Std + 2 Std + 2 Tris + 1xSSC Tris + 1xSSCStd buff + Std buff + 5% PEG + 5% PEG + 30% Form. 30% Form. D Std + 4Std + 4 25% ACN + 25% ACN + Std buff + Std buff + MES + 20% MES + 20%10% PEG + 10% PEG + PEG + 10% PEG + 10% 5% Form. 5% Form. Form. Form. EStd + 6 Std + 6 Std buf. + Std buf. + 10% PEG + 10% PEG + 20% PEG + 20%PEG + 2xSSC + 5% 2xSSC + 5% 10% Form. 10% Form. Form. Form. F Std + 8Std + 8 Std buf. + Std buf. + 10% Form. + 10% Form. + 10% Form. 10%Form. 2xSSC 2xSSC

“Graft” concentration refers to the concentration of surface boundoligos. Spot counts for each of the hybridization conditions weretabulated, whereby higher counts indicated more effective hybridizationbuffer formulations as shown in FIG. 4. Table 1 provides the buffercompositions and immobilized oligonucleotide concentrations for eachreaction seen in FIG. 4, with columns 10-21 in Table 1 correspondingwith columns 10-21 of FIG. 4, and rows B-F corresponding to row B-F ofFIG. 4.

Amplifying the hybridized target nucleic acid with surface bound nucleicacid. Following hybridization, the target nucleic acids were amplifiedto quantify hybridization effectiveness. Rolling circle amplification(RCA) was performed using amplification mixes with Bst according tomanufacturer's instructions (New England Biolabs®). The amplifiedcolonies of target nucleic acids were further amplified using a RCA/PCRamplification strategy, whereby PCR cycles were performed on the RCAmultimer nanoball to improve the detection sensitivity of the assay andmore stringently quantify hybridized library.

The resulting surface amplified products were again stained withintercalating DNA stains and imaged to verify hybridization specificityand effectiveness (See FIG. 5). Table 1 provides the buffer compositionsand immobilized oligonucleotide concentrations for each reaction seen inFIG. 5, with columns 10-21 in Table 1 corresponding with columns 10-21of FIG. 5, and rows B-F corresponding to row B-F of FIG. 5.

Analysis of Hybridization Buffers and conditions. Hybridizationconditions were evaluated based on the correlation of maximum spotcounts from FIG. 3, FIG. 4, and FIG. 5. Hybridization buffer C10, D18,and E21 showed the highest spot count, as compared to the negativecontrols provided in F10 and F11 in which water, instead ofhybridization buffer, was used. in FIG. 4. This result was validated inFIG. 5 after amplification.

Example 3

In this example, the non-specific binding of cyanine 3 dye (Cy3)-labeledmolecules was measured on the low non-specific binding surfacesdisclosed herein. In independent non-specific binding assays, 1 uMlabeled Cy3 dCTP (GE Amersham), 1 uM Cy5 dGTP dye (Jena Biosciences), 10uM Aminoallyl-dUTP—ATTO-647N (Jena Biosciences), 10 uMAminoallyl-dUTP—ATTO-Rho11 (Jena Biosciences), 10 μMAminoallyl-dUTP—ATTO-Rho11 (Jena Biosciences), 10 μM cCTP—Cy3.5 (GEAmersham), and 10 μM 7-Propargylamino-7-deaza-dGTP—Cy3 (JenaBiosciences) were incubated individually on the low non-specific bindingsurfaces described in Example 2 (Glass substrates treated withSilane-PEG5K, Nanocs) at 37° C. for 15 minutes in a 384 well plateformat. Each well was rinsed 2-3× with 50 μl deionized RNase/DNase Freewater and 2-3× with 25 mM ACES buffer pH 7.4. The 384 well plates wereimaged at single molecule resolution on an Olympus IX83 microscope(Olympus Corp., Center Valley, Pa.) with TIRF objective (100×, 1.4 NA,Olympus), a sCMOS camera (Zyla 4.2, Andor), an illumination source withexcitation wavelengths of 532 nm or 635 nm. Dichroic mirrors werepurchased from Semrock (IDEX Health & Science, LLC, Rochester, N.Y.),e.g., 405, 488, 532, or 633 nm dichroic reflectors/beamsplitters, andband pass filters were chosen as 532 LP or 645 LP concordant with theappropriate excitation wavelength. 5.

The imaging set-up enabled the visualization of single dye moleculesbound to the substrates. Individual fluorescent spots were counted andthe total spot numbers were divided by the respective area of the ROI.For example, with a 100× objective and Andor sCMOS camera, which has apixel size of 6.5 microns, it is possible to calculate the area of aregion of interest (ROI).

A low non-specific binding of the dye molecules above of less than orequal to about 0.50 molecules per μm² was observed. Some non-specificbinding of the dye molecules of less than or equal to 0.25 molecules perμm² was observed.

Example 4

A nucleic acid sequencing reaction is performed using the workflowprovided in FIG. 2 using the disclosed hybridization compositions andmethods from Example 1 and Example 2 on the surfaces used in Examples1-3. In this non-limiting example, the processing times that areachieved are also provided in FIG. 2.

Example 5: Preparation of Multivalent Binding Composition

One type of multi-armed substrate, as shown in FIG. 16A was made byreacting propargylamine dNTPs with Biotin-PEG-NHS. This aqueous reactionwas driven to completion and purified; resulting in a pureBiotin-PEG-dNTP species. In separate reactions, several different PEGlengths were used, corresponding to average molecular weights varyingfrom 1K Da to 20K Da. The Biotin-PEG-dNTP species were mixed with eitherfreshly prepared or commercially-sourced dye-labeled streptavidin (SA)using a Dye:SA ratio of 3-5:1. Mixing of Biotin-PEG-dNTP withdye-labeled streptavidin was done in the presence of excessbiotin-PEG-dNTP to ensure saturation of the biotin binding sites on eachstreptavidin tetramer. Complete complexes were purified away from excessbiotin-PEG-dNTP by size exclusion chromatography. Each nucleotide typewas conjugated and purified separately, then mixed together to create afour-base mix for sequencing.

Another type of multi-armed substrate as shown in FIG. 16A was made in asingle pot by reacting multi-arm PEG NHS with excess Dye-NH2 andpropargylamine dNTPs. Various multi-arm PEG NHS variants were usedranging from 4-16 arms and ranging in molecular weight from 5K Da to 40KDa. After reacting, excess small molecule dye and dNTP were removed bysize exclusion chromatography. Each nucleotide type was conjugated andpurified independently then mixed together to create a four-base mix forsequencing.

Class II substrates as shown in FIG. 16B were made using one potreactions to simultaneously conjugate dye and dNTP. Alkyne-PEG-NHS wasreacted with excess propargylamine dNTP. This product (Alkyne-PEG-dNTP)was then purified to homogeneity by chromatography. Multiple PEG lengthswere used, with average molecular weights varying between 1K Da and 20KDa. Dendrimer cores containing a variable, discrete number (12, 24, 48,96) of azide conjugation sites were used. Conjugation of Alkyne-Dye andAlkyne-PEG-dNTP to the dendrimer core occurred in a one pot reactioncontaining excess dye and dNTP species via copper-mediated clickchemistry. After reacting, excess small molecule dye and dNTP wereremoved by size exclusion chromatography. Each nucleotide type wasconjugated and purified independently then mixed together to create afour-base mix for sequencing. We note that this scheme allows the readysubstitution of alternative cores, such as dextrans, other polymers,proteins, etc.

Class III polymer-nucleotide conjugates as shown in FIG. 16C wereconstructed by reacting 4- or 8-arm PEG NHS with a saturating mixture ofbiotin and propargylamine dNTP. This reaction was then purified by sizeexclusion chromatography. The result of this reaction was a multi-armPEG containing a discrete distribution of biotin and nucleotides. Thisheterogeneous population was then reacted with dye-labeled streptavidinand purified by size exclusion chromatography. Each nucleotide type wasconjugated and purified independently then mixed together to create afour-base mix for sequencing. We note that the distribution of biotinand nucleotide is tunable by the input ratio of Biotin-NH2 topropargylamine dNTP.

Example 6: Detection of Ternary Complex

Binding reactions using the multivalent binding composition having PEGpolymer-nucleotide conjugates were analyzed to detect possible formationof ternary binding complex, and the fluorescence images of the varioussteps are illustrated in FIGS. 18A-18J. In FIG. 18A, red and greenfluorescent images post exposure of DNA rolling circle application (RCA)templates (G and A first base) to 500 nM base labeled nucleotides (A-Cy3and G-Cy5) in exposure buffer containing 20 nM Klenow polymerase and 2.5mM Sr+2. Multivalent PEG-substrate compositions were prepared usingvarying ratios of 4-armed PEG-amine (4ArmPEG-NH), biotin-PEG-amine(Biotin-PEG-NH), and nucleotide (Nuc) as follows: Samples PB1 and PB5,4ArmPEG-NH:Biotin-PEG-NH:Nuc=0.25:1:0.5; Sample PB2,4ArmPEG-NH:Biotin-PEG-NH:Nuc=0.125:0.5:0.25; Sample PB3,4ArmPEG-NH:Biotin-PEG-NH:Nuc=0.25:1:0.5. Images were collected afterwashing with imaging buffer with the same composition as the exposurebuffer but containing no nucleotides or polymerase.

Contrast was scaled to maximize visualization of the dimmest signals,but no signals persisted following washing with imaging buffer (FIG.18A, inset). In FIGS. 18B-18E, the fluorescence images showingmultivalent PEG-nucleotide (base-labeled) ligands at 500 nM after mixingin the exposure buffer and imaging in the imaging buffer as above (FIG.18B: PB1; FIG. 18C: PB2; FIG. 18D: PB3; FIG. 18E: PB5). FIG. 18F:fluorescence image showing multivalent PEG-nucleotide (base-labeled)ligand PB5 at 2.5 uM after mixing in the exposure buffer and imaging inthe imaging buffer as above. In FIGS. 18G-18I, the fluorescence imagesshowing further base discrimination by exposure of multivalent ligandsto inactive mutants of Klenow polymerase (FIG. 18G: D882H; FIG. 18H:D882E; FIG. 18I: D882A, and the wild type Klenow (control) enzyme isshown in FIG. 18J).

Using multivalent ligands formulations, the base discrimination can beenabled by providing polymerase-ligand interactions having increasedavidity. In addition, it is shown that increased concentration ofmultivalent ligands can generate higher signals, as well as variousKlenow mutations that knock out catalytic activity, and can be used foravidity-based sequencing.

Example 7: Sequencing of Target Nucleic Acid Molecules Using TernaryComplexes

In order to demonstrate sequencing based on multivalent ligandreporters, 4 known templates were amplified using RCA methods on a lowbinding substrate. Successive cycles were exposed to exposure buffercontaining 20 nM Klenow polymerase and 2.5 mM Sr+2 and washed withimaging buffer and imaged. After imaging, the substrates were washedwith wash buffer (EDTA and high salt) and blocked nucleotides were addedto proceed to the next base. The cycle was repeated for 5 cycles. Spotswere detected using standard imaging processing and spot detection andthe sequences were called using a two-color green and red scheme (G-Cy3and A-Cy5) to identify the templates being cycled. As shown in FIG. 19Aand FIG. 19B, multivalent ligands are able to provide basediscrimination through all 5 sequencing cycles.

Example 8: Control of Nucleotide Dissociation from Ternary Complex

Ternary complexes are prepared and imaged as in Example 6. The complexesare imaged over varying lengths of time to demonstrate the persistenceof the ternary complex, e.g., as long as 60 seconds. After a length oftime, the complexes are washed with a buffer identical to the bufferused for the formation of the complexes, only lacking any divalentcation, e.g., 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 0.016% TritonX100 (without SrOAc), or, in another example, the complexes are washedwith a buffer identical to the buffer used for the formation of thecomplexes, which contains a chelating agent but otherwise lacks anydivalent cation, e.g., 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl,0.016% Triton X100 (without SrOAc), with 100 nm-100 mM EDTA. Thefluorescence from the complexes is observed over time allowingobservation and quantitation of the dissociation of the ternarycomplexes. A representative time course of this dissolution is shown inFIG. 17.

Example 9: Extension of Target Nucleic Acid Complementary Sequence

After preparing, imaging, and dissociating ternary complexes as inExample 8, a deblocking solution is flowed into the chamber containingthe bound DNA molecules, sufficient to remove the blocking moiety, suchas an O-azidomethyl group, an O-alkyl hydroxylamino group, or an O-aminogroup, from the 3′ end of the elongating DNA strand. Either following orconcurrently with this, an extension solution is flowed into the chambercontaining the bound DNA molecules. The extension solution contains abuffer, a divalent cation sufficient to support polymerase activity, anactive polymerase, and an appropriate amount of all four nucleotides,where the nucleotides are blocked such that they are incapable ofsupporting further elongation after the addition of a single nucleotideto the elongating DNA strand, such as by incorporation of a3′-O-azidomnethyl group, a 3′-O-alkyl hydroxylamino group, or a3′-O-amino group. The elongating strand is thus extended by one and onlyone base, and the binding of catalytically inactive polymerase andmultivalent binding substrate can be used to call the next base in thecycle.

In another example, the nucleotides attached to the multivalentsubstrate may be attached through a labile bond, such that a buffer maybe flowed into the chamber containing the bound DNA molecules containinga divalent cation or other cofactor sufficient to render the polymerasecatalytically active. Prior to, after, or concurrently with this,conditions may be provided that are sufficient to cleave the base fromthe multivalent substrate such that it may be incorporated into theelongating strand. This cleavage and incorporation results in thedissociation of the label and the polymer backbone of the multivalentsubstrate while extending the elongating DNA strand by exactly one base.Washing to remove used polymer backbone is carried out, and newmultivalent substrate is flowed into the chamber containing the boundDNA molecules, allowing the new base to be called as in Example 5.

Example 10. Use of Polymer-Nucleotide Conjugates with Various Lengths ofPEG Branch

The polymer-nucleotide conjugates having varying PEG arm lengthsdescribed in Example 7 were subjected to a single sequencing cycle andimaged as described in Example 5. As shown in FIGS. 20A-20G, increasingthe length of the PEG branches led to increased signal up to a lengthcorresponding to an apparent average PEG MW of 5K Da (FIGS. 20A-20D).The use of longer PEG arms than this led to decreases in thefluorescence signal for both Cy3-A and Cy5-G (FIG. 20E-20G).Quantitative measurements of signal intensity are shown graphically inFIG. 21.

Example 11: Enhancement of Multivalent Substrate Binding by Addition ofDetergent

Multivalent substrates were prepared and assembled into bindingcomplexes in the presence and absence of detergent: one set using 10 mMTris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5 mM SroAc, 0% TritonX100(Condition A), and one set using 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mMNaCl, 5 mM SroAc, 0.016% Triton X100. FIG. 22 shows normalizedfluorescence from these multivalent substrates bound to DNA clusters,with the substrate complexes formed in the presence (condition B) ofTriton-X100 (0.016%) showing clearly enhanced fluorescence intensity.

Example 12. Evaluation of Multivalent Substrate Binding Time Courses

Multivalent substrates were prepared and assembled into bindingcomplexes as in Example 6. Complexes were also formed under identicalbuffer conditions using free labeled nucleotides. Complexes were imagedover the course of 60 min. to characterize the persistence time of thecomplexes. FIGS. 23A-23B shows representative results. Multivalentbinding complexes are stable over timescales of >60 minutes (FIG. 23B)while labeled free nucleotides dissociate in less than one minute (FIG.23A).

While preferred embodiments of the compositions and methods disclosedherein have been shown and described herein, it will be obvious to thoseskilled in the art that such embodiments are provided by way of exampleonly. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the present disclosure.It should be understood that various alternatives to the embodiments ofthe methods and compositions described herein may be employed in anycombination in practicing the methods and compositions of the presentdisclosure.

1.-20. (canceled)
 21. A method for hybridizing a target nucleic acidmolecule to a nucleic acid molecule coupled to a hydrophilic polymercoating layer, the method comprising: (a) providing a surface comprisingthe hydrophilic polymer coating layer coupled thereto; (b) providing atleast one nucleic acid molecule that is coupled to the hydrophilicpolymer coating layer, wherein the hydrophilic polymer coating layer hasa water contact angle of less than 45 degrees; and (c) bringing the atleast one nucleic acid molecule coupled to the hydrophilic polymercoating layer into contact with a hybridizing composition comprising atarget nucleic acid molecule at a concentration of one nanomolar or lessunder conditions sufficient for said target nucleic acid molecule tohybridize to the at least one nucleic acid molecule coupled to thehydrophilic polymer coating layer in 30 minutes or less.
 22. The methodof claim 21, wherein said conditions are maintained at a temperaturethat is from about 30 degrees Celsius to 70 degrees Celsius.
 23. Themethod of claim 21, wherein said hydrophilic polymer coating layercomprises a plurality of said at least one nucleic acid molecules with asubstantially uniform surface density.
 24. The method of claim 21,further comprising performing a nucleotide binding reaction on saidsurface between said at least one nucleic acid molecule and said targetnucleic acid molecule.
 25. The method of claim 21, wherein the targetnucleic acid molecule is present in the hybridizing composition at aconcentration of 0.50 nanomolar or less.
 26. The method of claim 21,wherein the target nucleic acid molecule is present in the hybridizingcomposition at a concentration of 250 picomolar or less.
 27. The methodof claim 21, wherein the target nucleic acid molecule is present in thehybridizing composition at a concentration of 100 picomolar or less. 28.The method of claim 21, wherein bringing the at least one nucleic acidmolecule coupled to the hydrophilic polymer coating layer into contactwith the hybridization composition is performed for a time period ofless than 30 minutes.
 29. The method of claim 21, further comprisinghybridizing the target nucleic acid molecule to the at least one nucleicmolecule coupled to the hydrophilic polymer coating layer at ahybridization efficiency that is increased as compared to a comparablehybridization reaction performed for 120 minutes at 90 degrees Celsiusfor 5 minutes followed by cooling for 120 minutes to reach a finaltemperature of 37 degrees Celsius in a buffer comprising saline-sodiumcitrate.
 30. The method of claim 21, further comprising hybridizing thetarget nucleic acid molecule to the at least one nucleic acid moleculewith a hybridization stringency of at least 80%.
 31. The method of claim21, wherein the hydrophilic polymer coating layer exhibits a level ofnon-specific Cyanine 3 dye absorption of less than about 0.25 moleculesper square micrometer.
 32. The method of claim 21, wherein thehybridization composition further comprises: (a) at least one organicsolvent that is polar and aprotic; and (b) a pH buffer.
 33. The methodof claim 32, wherein the at least one organic solvent has a dielectricconstant of no greater than about 115 as measured at 68 degreesFahrenheit.
 34. The method of claim 32, wherein the at least one organicsolvent comprises at least one functional group selected from hydroxy,nitrile, lactone, sulfone, sulfite, and carbonate.
 35. The method ofclaim 34, wherein the at least one organic solvent comprises formamide.36. The method of claim 32, wherein the at least one organic solvent ismiscible with water.
 37. The method of claim 32, wherein the at leastone organic solvent is at least about 5% by volume based on the totalvolume of the hybridizing composition.
 38. The method of claim 37,wherein the at least one organic solvent is at most about 95% by volumebased on the total volume of the hybridizing composition.
 39. The methodof claim 32, wherein the pH buffer is at most about 90% by volume of thetotal volume of the hybridizing composition.
 40. The method of claim 32,wherein the pH buffer comprises 2-(N-morpholino)ethanesulfonic acid,acetonitrile, 3-(N-morpholino)propanesulfonic acid, methanol, or acombination thereof.
 41. The method of claim 32, wherein the pH bufferfurther comprises a second organic solvent.
 42. The method of claim 32,wherein the pH buffer is present in the hybridizing composition in anamount that is effective to maintain the pH of the hybridizingcomposition in a range of about 3 to about
 10. 43. The method of claim21, wherein the hybridizing composition further comprises a molecularcrowding agent.
 44. The method of claim 43, wherein the molecularcrowding agent is selected from the group consisting of polyethyleneglycol, dextran, hydroxypropyl methyl cellulose, hydroxyethyl methylcellulose, hydroxybutyl methyl cellulose, hydroxypropyl cellulose,methyl cellulose, and hydroxyl methyl cellulose, and any combinationthereof.
 45. The method of claim 44, wherein the molecular crowdingagent is polyethylene glycol.
 46. The method of claim 45, wherein themolecular crowding agent has a molecular weight in the range of about5,000 to 40,000 Daltons.
 47. The method of claim 43, wherein an amountof the molecular crowding agent is at least about 5% by volume based onthe total volume of the hybridizing composition.
 48. The method of claim47, wherein an amount of the molecular crowding agent at most about 50%by volume based on the total volume of the hybridizing composition. 49.The method of claim 21, wherein the at least one nucleic acid moleculecoupled to the hydrophilic polymer coating layer is coupled to thehydrophilic polymer coating layer through covalent bonding.
 50. Themethod of claim 21, wherein the hydrophilic polymer coating layercomprises at least one dendrimer.